WO2022104071A2 - Agents thérapeutiques ciblant la signalisation de la famille bêta du facteur de croissance transformant - Google Patents

Agents thérapeutiques ciblant la signalisation de la famille bêta du facteur de croissance transformant Download PDF

Info

Publication number
WO2022104071A2
WO2022104071A2 PCT/US2021/059157 US2021059157W WO2022104071A2 WO 2022104071 A2 WO2022104071 A2 WO 2022104071A2 US 2021059157 W US2021059157 W US 2021059157W WO 2022104071 A2 WO2022104071 A2 WO 2022104071A2
Authority
WO
WIPO (PCT)
Prior art keywords
subject
agent
agents
combination
signaling
Prior art date
Application number
PCT/US2021/059157
Other languages
English (en)
Other versions
WO2022104071A9 (fr
WO2022104071A3 (fr
Inventor
Se-Jin Lee
Emily GERMAIN-LEE
Original Assignee
The Jackson Laboratory
University Of Connecticut
Connecticut Children's Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Jackson Laboratory, University Of Connecticut, Connecticut Children's Medical Center filed Critical The Jackson Laboratory
Priority to US18/036,704 priority Critical patent/US20230414711A1/en
Priority to EP21892877.8A priority patent/EP4243933A2/fr
Publication of WO2022104071A2 publication Critical patent/WO2022104071A2/fr
Publication of WO2022104071A3 publication Critical patent/WO2022104071A3/fr
Publication of WO2022104071A9 publication Critical patent/WO2022104071A9/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1719—Muscle proteins, e.g. myosin or actin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • MSTN Myostatin
  • TGF- ⁇ transforming growth factor- ⁇
  • Mice lacking MSTN exhibit dramatic increases in muscle mass throughout the body, with individual muscles growing to about twice the normal size.
  • MSTN appears to play two distinct roles in regulating MSTN also appears to play two distinct roles in regulating bone mineral density: one to limit differentiation of osteoblasts, which promote bone deposition, and another to promote the activity of osteoclasts, which promote bone resorption.
  • the sequence of MSTN has been highly conserved through evolution, with the mature MSTN peptide being identical in species as divergent as humans and turkeys, and the function of MSTN has also been conserved.
  • Targeted or naturally occurring mutations in MSTN have been shown to cause increased muscling in numerous species, including cattle, sheep, dogs, rabbits, rats, swine, goats, and humans.
  • MSTN neurotrophic factor-like protein
  • a wide range of indications including Duchenne and facioscapulohumeral muscular dystrophy, inclusion body myositis, spinal muscular atrophy, muscle atrophy following falls and hip fracture surgery, age- related sarcopenia, Charcot-Marie-Tooth disease, cachexia due to chronic obstructive pulmonary disease, end stage kidney disease, and cancer.
  • MSTN inhibitors have reached drug approval, and the effects of MSTN inhibition on bone growth are unknown.
  • the function of MSTN is partially redundant with that of another TGF- ⁇ family member, activin A.
  • MSTN and activin A signal through a complex of type 1 and type 2 receptors.
  • compositions comprising inhibitors of type 2 receptors (activin type 2 and/or TGF ⁇ type 2) or specific combinations of type 1 (activin type 1 and/or TGF ⁇ type I) and/or type 2 receptors, which respond to myostatin signaling by limiting skeletal muscle and bone growth.
  • Inhibiting specific combinations of receptors, as described herein provides key benefits over current methods, for example, those that target only type 1 receptors or those that target individual receptors.
  • Some aspects of the present disclosure provide a method of increasing muscle weight in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ALK4 and/or ALK5 signaling in the subject.
  • aspects of the present disclosure provide a method of reducing body fat content in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ALK4 and/or ALK5 signaling in the subject.
  • a method of improving glucose metabolism in a subject comprising administering to the subject an agent or a combination of agents that inhibit(s) ALK4 and/or ALK5 signaling in the subject.
  • a single agent is administered.
  • the single agent may inhibit, for example, ALK4 and ALK5 signaling (e.g., may bind to both ALK4 and ALK5).
  • the single agent may specifically inhibit ALK4 and ALK5 signaling.
  • two (or more, e.g., three or four) agents are administered.
  • one agent may inhibit (e.g., specifically inhibit) ALK4 signaling, while another agent may inhibit ALK5 signaling (e.g., specifically inhibit).
  • Non-limiting examples of such agents include, antibodies, soluble receptors, small molecules, and other non-peptide molecules, such as antisense oligonucleotides (ASOs), RNA interference (RNAi) molecules, and programmable-nuclease-based gene editing systems.
  • ASOs antisense oligonucleotides
  • RNAi RNA interference
  • programmable-nuclease-based gene editing systems include, antibodies, soluble receptors, small molecules, and other non-peptide molecules, such as antisense oligonucleotides (ASOs), RNA interference (RNAi) molecules, and programmable-nuclease-based gene editing systems.
  • ASOs antisense oligonucleotides
  • RNAi RNA interference
  • the agent or combination of agents is/are administered to the subject in an effective amount to (directly or indirectly) increase muscle weight (also referred to as muscle mass) in the subject by at least 40% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • a control may be administration of a placebo (e.g., saline) or baseline (e.g., muscle weight within 24 hours prior to administration of the one or more agent(s))).
  • the agent or combination of agents inhibit(s) ALK4 and/or ALK5 signaling by binding to (e.g., specifically binding to) ALK4 and/or ALK5.
  • the agent or combination of agents inhibit(s) ALK4 and/or ALK5 signaling specifically in myofibers of the subject.
  • the agent(s) may be selected from ASOs, RNAi molecules (e.g., shRNA, siRNA, or miRNA), and programmable nuclease- based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) that specifically target ALK4 and/or ALK5.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 50% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • Some aspects of the present disclosure provide a method of increasing muscle weight in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2A and ALK5 signaling in the subject.
  • aspects of the present disclosure provide a method of reducing body fat content in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2A and ALK5 signaling in the subject.
  • Yet other aspects of the present disclosure provide a method of improving glucose metabolism in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2A and ALK5 signaling in the subject.
  • a single agent is administered.
  • the single agent may inhibit, for example, ACVR2A and ALK5 signaling (e.g., may bind to both ACVR2A and ALK5).
  • the single agent may specifically inhibit ACVR2A and ALK5 signaling.
  • two (or more, e.g., three or four) agents are administered.
  • one agent may inhibit (e.g., specifically inhibit) ACVR2A signaling, while another agent may inhibit (e.g., specifically inhibit) ALK5 signaling.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents inhibit(s) ACVR2A and ALK5 signaling by binding to ACVR2A and ALK5.
  • the agent or combination of agents inhibit(s) ACVR2A and ALK5 signaling specifically in myofibers of the subject.
  • the agent(s) may be selected from ASOs, RNAi molecules (e.g., shRNA, siRNA, or miRNA), and programmable nuclease-based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) that specifically target ACVR2A and/or ALK5.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25% at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%relative to a control or baseline.
  • Some aspects of the present disclosure provide a method of increasing muscle weight in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2B and ALK5 signaling in the subject.
  • aspects of the present disclosure provide a method of reducing body fat content in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2B and ALK5 signaling in the subject.
  • Yet other aspects of the present disclosure provide a method of improving glucose metabolism in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2B and ALK5 signaling in the subject.
  • a single agent is administered.
  • the single agent may inhibit, for example, ACVR2B and ALK5 signaling (e.g., may bind to both ACVR2B and ALK5).
  • the single agent may specifically inhibit ACVR2B and ALK5 signaling.
  • two (or more, e.g., three or four) agents are administered.
  • one agent may inhibit (e.g., specifically inhibit) ACVR2B signaling, while another agent may inhibit (e.g., specifically inhibit) ALK5 signaling.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase muscle weight in the subject by at least 40% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents inhibit(s) ACVR2B and ALK5 signaling by binding to ACVR2B and ALK5.
  • the agent or combination of agents inhibit(s) ACVR2B and ALK5 signaling specifically in myofibers of the subject.
  • the agent(s) may be selected from ASOs, RNAi molecules (e.g., shRNA, siRNA, or miRNA), and programmable nuclease- based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) that specifically target ACVR2B and/or ALK5.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase tricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase quadricep muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase gastrocnemius/plantaris muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase pectoralis muscle weight in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase pectoralis muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits (e.g., specifically inhibits) ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits (e.g., specifically inhibits) ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase pectoralis muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • Some aspects of the present disclosure provide a method of increasing muscle weight in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) type I and/or type II receptor signaling in myofibers of the subject.
  • aspects of the present disclosure provide a method of reducing body fat content in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) type I and/or type II receptor signaling in myofibers of the subject.
  • Yet other aspects of the present disclosure provide a method of improving glucose metabolism in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) type I and/or type II receptor signaling in myofibers of the subject.
  • the type I receptor is selected from the group consisting of ALK4 and ALK5.
  • the agent or combination of agents inhibit(s) ALK4.
  • the agent or combination of agents inhibit(s) ALK5.
  • the agent or combination of agents inhibit(s) ALK4 and ALK5.
  • the agent or combination of agents that inhibit(s) type I receptor signaling binds to ALK4, ALK5 or both ALK4 and ALK5.
  • the type II receptor is selected from the group consisting of ACVR2A, ACVR2B, and TGF ⁇ RII.
  • the agent or combination of agents inhibit(s) ACVR2A.
  • the agent or combination of agents inhibit(s) ACVR2B.
  • the agent or combination of agents inhibit(s) TGF ⁇ RII.
  • the agent or combination of agents that inhibit(s) type I receptor signaling binds to ACVR2A, ACVR2B, TGF ⁇ RII, or any combination of two or three of the foregoing.
  • the agent or combination of agents inhibit(s) ALK4 and ALK5 signaling.
  • the agent or combination of agents inhibit(s) ACVR2A and ACVR2B signaling.
  • Some aspects of the present disclosure provide a method of increasing muscle weight in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling in the subject.
  • aspects of the present disclosure provide a method of reducing body fat content in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling in the subject.
  • Yet other aspects of the present disclosure provide a method improving glucose metabolism in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling in the subject.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase muscle weight in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, II and/or III signaling by binding to (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3.
  • the agent or combination of agents bind(s) to TGF ⁇ RII.
  • the agent or combination of agents bind(s) to TGF ⁇ 1.
  • the agent or combination of agents bind(s) to TGF ⁇ 2.
  • the agent or combination of agents bind(s) to TGF ⁇ 3.
  • the method further comprises administering to the subject an agent or a combination of agents that inhibit(s) type I receptor signaling in the subject. In some embodiments, the method further comprises administering to the subject an agent or a combination of agents that inhibit(s) type II receptor signaling in the subject. In some embodiments, the method further comprises administering to the subject an agent or a combination of agents that inhibit(s) type I receptor signaling and activin A type II receptor signaling in the subject.
  • the type I receptor is selected from the group consisting of ALK4 and ALK5. In some embodiments, the type I receptor is ALK4. In some embodiments, the type I receptor is ALK5. In some embodiments, the type II receptor is selected from the group consisting of ACVR2A, ACVR2B, and TGF ⁇ RII. In some embodiments, the type II receptor is ACVR2A. In some embodiments, the type II receptor is ACVR2B. In some embodiments, the type II receptor is TGF ⁇ RII.
  • the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII and one or more of the following pairs of receptors: (a) ALK4 and ALK5; (b) ACVR2A and AVCR2B; (c) ALK4 and ACVR2A; (d) ALK4 and ACVR2B; (e) ALK5 and ACVR2A; and (f) ALK5 and ACRV2B.
  • the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4 and ALK5.
  • the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ACVR2A and AVCR2B. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4 and ACVR2A. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4 and ACVR2B. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5 and ACVR2A. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5 and ACRV2B.
  • Some aspects of the present disclosure provide a method of increasing bone mineral density, bone volume, and/or bone density in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ALK4 and/or ALK5 signaling in the subject.
  • bone mineral density, bone volume, and/or bone density is increased in the hip, lumbar spine, forearm or whole body of the subject.
  • a single agent is administered.
  • the single agent may inhibit, for example, ALK4 and ALK5 signaling (e.g., may bind to both ALK4 and ALK5).
  • the single agent may specifically inhibit ALK4 and ALK5 signaling.
  • two (or more, e.g., three or four) agents are administered.
  • one agent may inhibit (e.g., specifically inhibit) ALK4 signaling, while another agent may inhibit ALK5 signaling (e.g., specifically inhibit).
  • Non-limiting examples of such agents include, antibodies, soluble receptors, small molecules, and other non-peptide molecules, such as antisense oligonucleotides (ASOs), RNA interference (RNAi) molecules, and programmable-nuclease-based gene editing systems.
  • ASOs antisense oligonucleotides
  • RNAi RNA interference
  • the agent or combination of agents is/are administered to the subject in an effective amount to (directly or indirectly) increase bone mineral density, bone volume, and/or bone density in the subject by at least 40% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • a control may be administration of a placebo (e.g., saline) or baseline (e.g., bone mineral density within 24 hours prior to administration of the one or more agent(s))).
  • the agent or combination of agents inhibit(s) ALK4 and/or ALK5 signaling by binding to (e.g., specifically binding to) ALK4 and/or ALK5.
  • the agent or combination of agents inhibit(s) ALK4 and/or ALK5 signaling specifically in osteoblasts of the subject.
  • the agent(s) may be selected from ASOs, RNAi molecules (e.g., shRNA, siRNA, or miRNA), and programmable nuclease- based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) that specifically target ALK4 and/or ALK5.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase total body bone mineral density by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density by at least 3%, at least 4%, or at least 5% at a site selected from the group consisting of lumbar spine, radius, ulna, and pelvis, relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 50% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ALK4 and/or ALK5 signaling may be administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ALK4 signaling (e.g., by binding to ALK4) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • Some aspects of the present disclosure provide a method of increasing bone mineral density, bone volume, and/or bone density in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) ACVR2A and ALK5 signaling in the subject.
  • a single agent is administered.
  • the single agent may inhibit, for example, ACVR2A and ALK5 signaling (e.g., may bind to both ACVR2A and ALK5).
  • the single agent may specifically inhibit ACVR2A and ALK5 signaling.
  • two (or more, e.g., three or four) agents are administered.
  • one agent may inhibit (e.g., specifically inhibit) ACVR2A signaling, while another agent may inhibit (e.g., specifically inhibit) ALK5 signaling.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents inhibit(s) ACVR2A and ALK5 signaling by binding to ACVR2A and ALK5.
  • the agent or combination of agents inhibit(s) ACVR2A and ALK5 signaling specifically in osteoblasts of the subject.
  • the agent(s) may be selected from ASOs, RNAi molecules (e.g., shRNA, siRNA, or miRNA), and programmable nuclease-based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) that specifically target ACVR2A and/or ALK5.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase total body bone mineral density by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density by at least 3%, at least 4%, or at least 5% at a site selected from the group consisting of lumbar spine, radius, ulna, and pelvis, relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25% at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2A and ALK5 signaling may be administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% relative to a control or baseline.
  • two agents one that inhibits ACVR2A signaling (e.g., by binding to ACVR2A) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%relative to a control or baseline.
  • a single agent is administered.
  • the single agent may inhibit, for example, ACVR2B and ALK5 signaling (e.g., may bind to both ACVR2B and ALK5).
  • the single agent may specifically inhibit ACVR2B and ALK5 signaling.
  • two (or more, e.g., three or four) agents are administered.
  • one agent may inhibit (e.g., specifically inhibit) ACVR2B signaling, while another agent may inhibit (e.g., specifically inhibit) ALK5 signaling.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 40% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents inhibit(s) ACVR2B and ALK5 signaling by binding to ACVR2B and ALK5.
  • the agent or combination of agents inhibit(s) ACVR2B and ALK5 signaling specifically in osteoblasts of the subject.
  • the agent(s) may be selected from ASOs, RNAi molecules (e.g., shRNA, siRNA, or miRNA), and programmable nuclease- based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) that specifically target ACVR2B and/or ALK5.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase total body bone mineral density by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density by at least 3%, at least 4%, or at least 5% at a site selected from the group consisting of lumbar spine, radius, ulna, and pelvis, relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase lumbar spine bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase radius and/or ulna bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase pelvis bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • the agent or combination of agents is/are administered to the subject in an effective amount to increase vertebrae bone mineral density in the subject by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • a single agent that inhibits (e.g., specifically inhibits) ACVR2B and ALK5 signaling may be administered to the subject in an effective amount to increase vertebrae bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • two agents one that inhibits (e.g., specifically inhibits) ACVR2B signaling (e.g., by binding to ACVR2B) and one that inhibits (e.g., specifically inhibits) ALK5 signaling (e.g., by binding to ALK5) may be administered to the subject in an effective amount to increase vertebrae bone mineral density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% relative to a control or baseline.
  • Some aspects of the present disclosure provide a method of increasing bone mineral density, bone volume, and/or bone density in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) type 1 and/or type 2 receptor signaling in osteoblasts of the subject.
  • the type 1 receptor is selected from the group consisting of ALK4 and ALK5.
  • the agent or combination of agents inhibit(s) ALK4.
  • the agent or combination of agents inhibit(s) ALK5.
  • the agent or combination of agents inhibit(s) ALK4 and ALK5.
  • the agent or combination of agents that inhibit(s) type 1 receptor signaling binds to ALK4, ALK5 or both ALK4 and ALK5.
  • the type 2 receptor is selected from the group consisting of ACVR2A, ACVR2B, and TGF ⁇ RII.
  • the agent or combination of agents inhibit(s) ACVR2A.
  • the agent or combination of agents inhibit(s) ACVR2B.
  • the agent or combination of agents inhibit(s) TGF ⁇ RII.
  • the agent or combination of agents that inhibit(s) type 1 receptor signaling binds to ACVR2A, ACVR2B, TGF ⁇ RII, or any combination of two or three of the foregoing.
  • the agent or combination of agents inhibit(s) ALK4 and ALK5 signaling.
  • the agent or combination of agents inhibit(s) ACVR2A and ACVR2B signaling.
  • Some aspects of the present disclosure provide a method of increasing muscle weight in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling in the subject.
  • aspects of the present disclosure provide a method of reducing body fat content in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling in the subject.
  • Yet other aspects of the present disclosure provide a method improving glucose metabolism in a subject, comprising administering to the subject an agent or a combination of agents that inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling in the subject.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase total body bone mineral density by at least 3%, at least 4%, or at least 5% relative to a control or baseline.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density by at least 3%, at least 4%, or at least 5% at a site selected from the group consisting of lumbar spine, radius, ulna, and pelvis, relative to a control or baseline.
  • the agent or a combination of agents is/are administered to the subject in an effective amount to increase bone mineral density, bone volume, and/or bone density in the subject by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% relative to a control or baseline.
  • the agent or combination of agents inhibit(s) (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, II and/or III signaling by binding to (a) TGF ⁇ RII and/or (b) TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3.
  • the agent or combination of agents bind(s) to TGF ⁇ RII.
  • the agent or combination of agents bind(s) to TGF ⁇ 1.
  • the agent or combination of agents bind(s) to TGF ⁇ 2.
  • the agent or combination of agents bind(s) to TGF ⁇ 3.
  • the method further comprises administering to the subject an agent or a combination of agents that inhibit(s) type 1 receptor signaling in the subject. In some embodiments, the method further comprises administering to the subject an agent or a combination of agents that inhibit(s) type 2 receptor signaling in the subject. In some embodiments, the method further comprises administering to the subject an agent or a combination of agents that inhibit(s) type 1 receptor signaling and activin A type 2 receptor signaling in the subject.
  • the type 1 receptor is selected from the group consisting of ALK4 and ALK5. In some embodiments, the type 1 receptor is ALK4. In some embodiments, the type 1 receptor is ALK5.
  • the type 2 receptor is selected from the group consisting of ACVR2A, ACVR2B, and TGF ⁇ RII. In some embodiments, the type 2 receptor is ACVR2A. In some embodiments, the type 2 receptor is ACVR2B. In some embodiments, the type 2 receptor is TGF ⁇ RII.
  • the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII and one or more of the following pairs of receptors: (a) ALK4 and ALK5; (b) ACVR2A and AVCR2B; (c) ALK4 and ACVR2A; (d) ALK4 and ACVR2B; (e) ALK5 and ACVR2A; and (f) ALK5 and ACRV2B.
  • the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4 and ALK5.
  • the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ACVR2A and AVCR2B. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4 and ACVR2A. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4 and ACVR2B. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5 and ACVR2A. In some embodiments, the method comprises administering to the subject an agent or a combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5 and ACRV2B.
  • FIGs. 1A-1F Effect of targeting type II and type I receptors in myofibers on muscle weights.
  • FIG. 1A-1B Relative weights of pectoralis, triceps, quadriceps, and gastrocnemius/plantaris muscles in mice in which Acvr2 and/or Acvr2b (FIG. 1A) or Alk4 and/or Alk5 (FIG. 1B) were targeted. Numbers are expressed as percent increase/decrease relative to the same receptor genotypes but in the absence of Afy/7-Cre.
  • FIG. 1A-1B Relative weights of pectoralis, triceps, quadriceps, and gastrocnemius/plantaris muscles in mice in which Acvr2 and/or Acvr2b (FIG. 1A) or Alk4 and/or Alk5 (FIG. 1B) were targeted. Numbers are expressed as percent increase/decrease relative to the same receptor genotypes but in the absence of Afy/7-
  • FIG. 1C Gastrocnemius/plantaris muscle weights of individual wild type C57BL/6 and Mstn -I- mice or individual mice in which Acvr2/Acvr2b or Alk4/Alk5 were targeted in myofibers. Bars indicates mean values.
  • FIG. 1D Relative muscle weights of mice in which an individual type II receptor (Acvr2 or Acvr2b) was targeted along with an individual type I receptor (Alk4 or Alk5).
  • FIGs. 1E-1F Relative muscle weights of mice in which an individual type II or type I receptor was targeted along with Cfc1b (FIG. 1E) or Mstn (FIG. 1F).
  • FIGs. 1A-1B and FIGs. 1D-1F were calculations based on muscle weights shown in Tables 1-3, which also contain the numbers of mice in each group. a p ⁇ 0.001 vs. cre -; b p ⁇ 0.01 vs. cre -; c p ⁇ 0.05 vs. cre -; d p ⁇ 0.001 vs. Mstnfl/fl, cre +; e p ⁇ 0.01 vs. vs. Mstnfl/fl, cre +; f p ⁇ 0.05 vs. vs. Mstnfl/fl, cre +.
  • FIGs. 2A-2C Lack of effect of targeting Acvr2 and Acvr2b in myofibers on muscle regeneration following chemical injury.
  • FIG. 2A Distribution of myofiber cross-sectional areas (CSA), mean CSA (FIG. 2B), and number of Pax7+ cells (FIG. 2C) in Acvr2fl/fl, Acvr2b fl/fl mice with or without Myl1 -Cre either uninjured or 5 or 21 days post- injury.
  • FIG. 3 Total body fat content by DXA analysis, plasma leptin levels, fasting blood glucose levels, and fasting plasma insulin levels in one-year-old mice lacking MSTN and mice in which both type II receptors were targeted in myofibers. Numbers of mice in each group are shown underneath the bars.
  • FIGs. 4A-4C Effect of a high fat diet on Mstn -/- mice and mice in which Acvr2 and Acvr2b have been targeted in myofibers.
  • FIG. 4A Weight gain in male mice placed on a high fat diet starting at 12 weeks of age.
  • FIG. 4B Fasting blood glucose levels and
  • FIG. 4C glucose tolerance tests in 12-week-old male mice on standard diets or after placement on a high fat diet for 4 weeks.
  • the numbers of mice in each group are the same as shown in panel (FIG. 4A).
  • FIGs. 5A-5C Lack of bone effects of targeting Acvr2 and Acvr2b in myofibers.
  • FIG. 5B MicroCT images of femurs taken from these same mice at 16 weeks of age.
  • FIG. 5C Bone volume/total volume fraction, trabecular thickness, trabecular number), apparent density, and cortical thickness of femurs and L4 and L5 vertebrae determined by microCT analysis in these mice at 16 weeks of age. Numbers of mice in each group are shown underneath the bars.
  • FIGs. 6A-6F Effect of Fst mutant alleles on skeletal muscle.
  • FIG. 6A Weights of gastrocnemius muscles versus Fst RNA expression levels in mice carrying various combinations of Fst mutant alleles. Numbers are normalized to values for Fst +/+ muscles. RNA expression levels were measured by qPCR in 3 mice per group.
  • FIG. 6C Relative weights of pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius/plantaris (green) muscles in mice carrying various Fst mutant alleles with or without Myl1 -cre. Numbers are expressed as percent increase/decrease relative to Fst +/+ mice and were calculated from the data shown in Table 1.
  • FIG. 6D Relative weights of muscles of Fst fl/- mice in the absence (blue bars) or presence (orange bars) of Cdx2-cre. Numbers are expressed as percent increase/decrease relative to Fst +/+ mice.
  • FIG. 6E Distribution of fiber sizes in gastrocnemius muscles of F66 and Fst +/+ mice (upper panel) or Fst fl/- ; Cdx2-cre negative and positive mice (lower panel). Fiber diameters were measured in muscles isolated from 3 mice (250 fibers per mouse) and pooled for plotting.
  • FIGs. 7A-7B Fiber type shifts in Fst mutant mice.
  • FIGs. 7A-7C Lipid accumulation in Fst mutant mice.
  • FIG. 7A Oil Red O stains of gastrocnemius sections of Fst fl/- ; Cdx2-cre negative and positive mice.
  • FIGs. 8A-8C Effect of Fst mutant alleles on bone.
  • FIG. 8C Micro-CT analysis of femurs and humeri isolated from Fst fl/- ; Cdx2-cre negative and positive mice. F, females; M, males.
  • FIGs. 4A-4C numbers of mice per group are shown at bottom.
  • FIGs. 9A-9B numbers of mice per group are shown at bottom.
  • a p ⁇ 0.001, b p ⁇ 0.01, c p ⁇ 0.05 Representative micro-CT images.
  • FIGs 10A-10C Effect of targeting Mstn and Inhba in the posterior half of mice.
  • FIG. 10B Muscle weight increases in Mstn flox/flox ; Inhba flox/flox ; Cdx2-cre mice relative to ere negative control mice.
  • FIG. 10C Micro-CT analysis of humeri, femurs, and L5 vertebrae of Msln flox/flox ; Inhba flox/flox ; Cdx2-cre. a p ⁇ 0.001, b p ⁇ 0.01, c p ⁇ 0.05.
  • FIG. 11 shows Venn diagrams of the numbers of genes whose RNA expression levels are either up- or down-regulated in gastrocnemius muscles isolated from either Fst floxl- ; Cdx2- cre (relative to ere negative mice) or F66 mice (relative to wild-type mice).
  • FIG. 12 shows expression levels of Mstn RNA in various muscles isolated from Fst floxl- mice either positive or negative for Cdx2-cre. Expression levels in ere negative mice were arbitrarily set to one for each muscle group.
  • Myostatin is a secreted protein that is made by skeletal muscle, circulates in the blood, and acts to limit muscle growth. Signaling of myostatin and other activin-like ligands through TGF- ⁇ superfamily receptors, such as the type 1 receptors ALK4 and ALK5 and the type 2 receptors ACVR2A and ACVR2B, regulates numerous developmental pathways, including muscle and bone growth. As a result, the myostatin signaling pathway has been the focus of extensive drug development efforts for indications characterized by muscle loss.
  • muscle degenerative diseases muscle dystrophy
  • muscle wasting caused by chronic diseases e.g., cancer, AIDS, sepsis, COPD, kidney disease, heart disease
  • muscle loss due to disuse e.g., elderly, bedridden, and wheelchair-using individuals.
  • myostatin inhibitors that have been tested in clinical trials, many of which reached phase 2 or 3 clinical trials. Although these trials have documented increases in muscle mass as a result of treatment, the effects have been small, and no one has yet documented any clinically meaningful benefits from treatment. None of these myostatin inhibitors have reached drug approval.
  • myostatin In parallel with its effects on muscle growth, myostatin also limits bone density by inhibiting osteoblast differentiation and promoting osteoclast activity. Consequently, this signaling pathway is also the focus of research and development for treating indications characterized by bone loss, such as osteoporosis, Cushing’s syndrome, pituitary disorders, and hyperthyroidism, as well as bone loss due to inactivity or chronic diseases described above.
  • Current therapies for treating bone loss include calcium supplementation to promote new bone deposition, and alendronic acid (Fosamax) to inhibit bone resorption by osteoclasts. However, the effectiveness of these therapies is limited, with minimal success in replenishing lost bone.
  • MSTN and activin A share many key regulatory and signaling components.
  • the activities of both MSTN and activin A can be modulated extracellularly by naturally occurring inhibitory binding proteins, including follistatin and the follistatin-related protein, FSTL-3 or FLRG.
  • mice that express greater amounts of follistatin, which sequesters myostatin and prevents it from acting as a receptor agonist experience increased bone density than wild-type mice or mice in which the FST gene is not expressed in the bone microenvironment.
  • MSTN and activin A also appear to share receptor components.
  • MSTN binds initially to the type 2 receptors, ACVR2 and ACVR2B (also called ActRIIA and ActRIIB) followed by engagement of the type 1 receptors, ALK4 and ALK5.
  • ACVR2 and ACVR2B also called ActRIIA and ActRIIB
  • ALK4 and ALK5 genetic evidence supports a role for both ACVR2 and ACVR2B in mediating MSTN signaling, regulating muscle mass, and regulating bone mineral density in vivo.
  • mice expressing a truncated, dominant negative form of ACVR2B in skeletal muscle or carrying deletion mutations in Acvr2 and/or Acvr2b have significantly increased muscle mass.
  • the present disclosure provides, in some aspects, methods and compositions that utilize inhibitors that have a broader range of specificity (broader than just myostatin) while avoiding undesired effects in other tissues as a result of blocking the other signaling proteins.
  • Provided herein is an extensive analysis of the receptors that are used in skeletal muscle cells and osteoblasts by myostatin and activin A for signaling.
  • These ligands utilize a two-component system for signaling, with initial binding to a type 2 receptor (ACVR2A or ACVR2B) and subsequent engagement of a type 1 receptor (ALK4 or ALK5).
  • therapeutic agents and the use of therapeutic agents that inhibit (e.g., specifically inhibit) signaling through ALK5 and ACVR2.
  • therapeutic agents include an ACVR2/ALK5 hybrid decoy receptor (e.g., a heterodimer of the extracellular domains of each of these receptors fused to a scaffold, such as an immunoglobulin Fc domain) and a peptide or small molecule inhibitors capable of disrupting the ACVR2-ALK5 interaction.
  • the present disclosure provides, in some aspects, methods of administering inhibitors of type 1 receptor and/or type 2 receptor signaling to a subject, thereby reducing or blocking the activity of myostatin.
  • Myostatin is a protein that is produced by muscle cells and associates with type 1 receptors and/or type 2 receptors on cell surfaces.
  • the first step in myostatin signaling is the association of myostatin with a type 2 receptor, such as ACVR2A, ACVR2B, or TGF ⁇ RII.
  • a type 2 receptor such as ACVR2A, ACVR2B, or TGF ⁇ RII.
  • the type 2 receptor then associates with a type 1 receptor, forming a type 1 receptor/type 2 receptor complex.
  • This complex then activates transcription factors, such as SMAD2 and SMAD3, which modulate gene expression in the cell and ultimately inhibit growth. In immature muscle cells (myoblasts), these signals inhibit differentiation into mature muscle fibers.
  • Myostatin signaling also inhibits the activity of the protein kinase B (PKB), also known as Akt, a serine/threonine protein kinase that plays a key role in multiple cellular processes, including glucose metabolism, gene regulation, and cell proliferation.
  • PPKB protein kinase B
  • Akt protein kinase B
  • the activity of Akt promotes growth and/or proliferation of muscle cells, resulting in increased muscle weight in animals where myostatin signaling is inhibited.
  • myostatin signaling in bone-forming cells osteoblasts
  • myostatin signaling promotes the expression of several bone regulatory factors, such as sclerostin, DKK1, and RANKL, and suppresses other miRNAs involved in in osteoblast development. These combined effects prevent the differentiation of osteoblasts and, consequently, the formation of new bone.
  • type 1 receptor and/or type 2 receptor signaling is inhibited specifically in myofibers.
  • Myofibers also referred to as myocytes or muscle cells, are cells present in muscle tissue, including skeletal muscle, smooth muscle, and cardiac muscle. Targeted inhibition of myostatin signaling in myofibers thus promotes muscle growth without undesired side effects that may result from inhibition of type 1 receptor signaling and/or type 2 receptor signaling in other cell types or tissues.
  • type 1 receptor and/or type 2 receptor signaling is inhibited specifically in osteoblasts. Osteoblasts are cells present in bones that synthesize collagen, osteocalcin, and osteopontin, which together form the organic matrix of bone.
  • Targeted inhibition in myofibers and/or osteoblasts may be achieved, for example, by conditionally expressing agents such as antisense oligonucleotides (ASOs), RNAi molecules (e.g., shRNA, siRNA, or miRNA), and/or programmable nuclease-based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) in myofibers and/or osteoblasts.
  • agents such as antisense oligonucleotides (ASOs), RNAi molecules (e.g., shRNA, siRNA, or miRNA), and/or programmable nuclease-based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) in myofibers and/or osteoblasts.
  • Some aspects of the present disclosure provide methods of administering to a subject an agent or composition that inhibits signaling by receptors.
  • “Signaling,” as used herein, is the process by which information about the state of a biological system, such as the presence, absence, or amount of a substance, is detected and processed by a cell or organism.
  • a “receptor” is a protein that can detect and transduce a biological signal, such as the presence of a ligand or, more generally, associate with a ligand.
  • Receptors may be present outside of a cell (soluble receptors), attached to the exterior surface of a cell (extracellular receptors), embedded in the plasma membrane of a cell (transmembrane receptors), attached to the interior surface of a cell (intracellular receptors), or contained within the cytoplasm (cytoplasmic receptors and/or adaptor proteins). Receptors may contain multiple domains, located outside of the cell (extracellular domain), within the plasma membrane (transmembrane domain), and/or inside the cell (intracellular or cytoplasmic domain).
  • Receptors may associate with, and thereby detect the presence of, a ligand.
  • a “ligand” is a substance, such as a protein, that associates with a receptor or other biomolecule. Association between a ligand and receptor generally occurs via intramolecular forces such as hydrogen bonds, ionic bonds, and Van der Waals forces. Non-limiting examples of ligands include carbohydrates, lipids, small molecules, macromolecules, nucleic acids, peptides and neurotransmitters. Association between a ligand and receptor may result in a biological process that does not occur when the receptor is not associated with the ligand, such as signal transduction. As used herein, “signal transduction” refers to a process in which an extracellular signal is transmitted to the intracellular environment.
  • the extracellular signal may be the presence of a ligand, or the association of a ligand with a receptor.
  • Signal transduction may occur through phosphorylation, dephosphorylation, conformational changes, and/or other chemical modifications of the receptor following association of a ligand with a receptor. Such modifications may result in the association between the ligand-bound receptor and other receptors, and/or the association of other intracellular proteins with intracellular or transmembrane domains of the receptor.
  • compositions described herein may contain inhibitors of type 1 receptor or type 2 receptor signaling.
  • an “inhibitor” refers to an agent or a combination of agents that inhibit(s) signaling of a particular receptor.
  • An inhibitor may specifically inhibit signaling by a receptor.
  • Specific inhibition refers to the action of an agent that prevents (no measurable activity) or reduces (e.g., by at least 10% relative to a control) the function of a given protein, such as by preventing signal transduction by a protein association of the protein with a ligand, which occurs more frequently than the action of the same agent against other proteins.
  • An inhibitor may inhibit protein function (activity) by acting directly or indirectly on the protein, or by modifying (e.g., preventing) expression of the protein by modifying translation of the protein or transcription of the DNA/mRNA encoding the protein.
  • a reduction in protein activity may be a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (no measurable activity), relative to a control (e.g., protein activity in the absence of the inhibitor).
  • the inhibitor associates with (e.g., binds to) the receptor, preventing other ligands from associating with the same receptor (competitive inhibition), but this association does not trigger signal transduction, and thus the presence of the inhibitor reduces the signaling activity of the receptor. Inhibition may occur by specific binding.
  • Specific binding refers to the non-random association of an agent with a given protein, with the agent showing significantly less affinity for other proteins. Binding affinity between an agent a given protein may be quantified as the dissociation constant (K D ) of the interaction between the agent and protein using methods known in the art.
  • the binding affinity of an agent to one protein relative to another may be evaluated by measuring the K D values of the agent’ s interaction with each protein and comparing them using statistical methods that are known in the art (e.g., Student’s t-test, ANOVA, regression models).
  • An agent is said to bind to a given protein with significantly more affinity relative to another protein if 1) the K D of the agent’s interaction with the given protein is lower than the K D of the agent’s interaction with the other protein, and 2) a statistical test or model determines that the probability of the results being due to chance is less than 0.1, less than 0.05, or less than 0.01.
  • the inhibitor is a soluble receptor (or “decoy receptor”) that associates with (e.g., binds to) the same ligand with which the targeted receptor (e.g., type 1 and/or type 2 receptor) associates.
  • the soluble receptor As the soluble receptor is not associated with a cell, its association with ligand will not trigger signal transduction, but will reduce the concentration of ligand in an environment, such as the bloodstream, and thus reduce the frequency of signal transduction in cells due to the lower availability of ligand (indirect inhibition).
  • the soluble receptor comprises an extracellular domain of a type 1 and/or type 2 receptor.
  • the soluble receptor comprises an (at least one) extracellular domain of ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII.
  • the soluble receptor comprises the extracellular domains of ALK4 and ALK5. In some embodiments, the soluble receptor comprises the extracellular domains of ALK4 and ACVR2A. In some embodiments, the soluble receptor comprises the extracellular domains of ALK4 and ACVR2B. In some embodiments, the soluble receptor comprises the extracellular domains of ALK5 and ACVR2A. In some embodiments, the soluble receptor comprises the extracellular domains of ALK5 and ACVR2B. In some embodiments, the soluble receptor comprises the extracellular domains of ACVR2A and ACVR2B.
  • the soluble receptor comprises the extracellular domains of TGF ⁇ RII, ALK4, and ALK5. In some embodiments, the soluble receptor comprises the extracellular domains of TGF ⁇ RII, ALK4, and ACVR2A. In some embodiments, the soluble receptor comprises the extracellular domains of TGF ⁇ RII, ALK4, and ACVR2B. In some embodiments, the soluble receptor comprises the extracellular domains of TGF ⁇ RII, ALK5, and ACVR2A. In some embodiments, the soluble receptor comprises the extracellular domains of TGF ⁇ RII, ALK5, and ACVR2B. In some embodiments, the soluble receptor comprises the extracellular domains of TGF ⁇ RII, ACVR2A, and ACVR2B.
  • a soluble receptor may also comprise additional domains (e.g., antibody Fc domain) that promote its degradation, clearance from circulation, or sequestration of the receptor and bound ligand away from other cells. See, e.g., Puolakkainen et al. BMC Musculoskelet Disord. (2017). 18(l):20.
  • additional domains e.g., antibody Fc domain
  • Inhibitors in some embodiments, associate with domains of a receptor involved in signal transduction, such that the receptor may still associate with ligand but exhibit reduced signal transduction activity.
  • agents that inhibit type 1 and type 2 receptor signaling are provided throughout the present disclosure. See, e.g., “Methods of Inhibiting MSTN and/or Activin Signaling” below.
  • Inhibitors may reduce or prevent expression of a receptor in a cell, thereby reducing the frequency of signal transduction due to the lower availability of receptors on or in a cell, such as an osteoblast or muscle cell.
  • Non-limiting examples of inhibitors that act in this manner include antisense oligonucleotides, small interfering RNAs, short hairpin RNAs, microRNAs, and programmable nucleases (e.g., RNA-guided nucleases, TALENs, and ZFNs).
  • Inhibitors may specifically target an mRNA or genomic locus encoding a desired protein.
  • Specifically target refers to the non-random action of an agent against a particular target, such as an mRNA or genomic locus encoding a particular protein, with the agent showing significantly less action against other targets, such as an mRNAs or genomic loci encoding proteins other than the particular protein.
  • Action of an agent against a target may be quantified by measuring the change in DNA sequence, mutation frequency, mRNA transcription, mRNA abundance, and/or protein translation that result from the addition of the agent, using methods known in the art (e.g., PCR, qRT-PCR, western blotting).
  • the action of an agent against one target relative to another target may be evaluated by measuring the action of the agent against each target, and comparing the measurements using statistical methods that are known in the art (e.g., Student’s t-test, ANOVA, regression models).
  • An agent is said to have significantly more action against a given target relative to another target if 1) the magnitude of the action as measured is higher against the given target than another target, and 2) a statistical test or model determines that the probability of the results being due to chance is less than 0.1, less than 0.05, or less than 0.01.
  • an inhibitor is an antisense oligonucleotide (ASO).
  • ASO is a single-stranded DNA or RNA oligonucleotide that is complementary to a target sequence.
  • An oligonucleotide is complementary to a target sequence if the oligonucleotide binds to a nucleic acid comprising the target sequence, forming a nucleic acid that is at least partially double- stranded through hydrogen bonds between base pairs on the oligonucleotide and target sequence.
  • An oligonucleotide is most complementary to a sequence when the oligonucleotide comprises a sequence of bases that form canonical Watson-Crick base pairs (i.e., A-U, A-T, C-G) with the target sequence, in reverse order relative to the order of bases in the target sequence. Binding of an antisense oligonucleotide to an mRNA target can interfere with normal cellular processing of the mRNA, and therefore expression of the encoded protein, through multiple mechanisms.
  • nucleases of the ribonuclease H (RNase H or RNH) family hydrolyze the phosphodiester bond between nucleotides in the RNA component of a DNA/RNA hybrid nucleic acid, in which a single-stranded DNA sequence is hybridized with a single-stranded RNA sequence.
  • RNase H or RNH ribonuclease H
  • An mRNA bound by an antisense DNA oligonucleotide may thus be cleaved by RNase H, thereby preventing translation of the mRNA into an encoded protein.
  • ASOs may also modulate gene expression by interfering with the formation of a 5’ cap on mRNA, altering the splicing process (splice-switching), and hindering translation by ribosomes through steric hindrance. See, e.g., Rinaldi et al. Nat Rev Neurol. (2016). 14(1):9— 21.
  • an inhibitor is a small interfering RNA.
  • a small interfering RNA also known as short interfering RNA or silencing RNA, is a double- stranded RNA (dsRNA) that contains one strand that is complementary to a chosen target sequence (guide strand), and one strand that is complementary to the guide strand (passenger strand).
  • the protein Argonaute (Ago) associates with the dsRNA, after which the guide strand is integrated into the RNA-induced signaling complex (RISC), and the passenger strand is degraded.
  • RISC RNA-induced signaling complex
  • RISC may also inhibit the translation of mRNAs containing a sequence complementary to a guide RNA by preventing the addition of a 5’ cap, removing the 3’ poly(A) tail, or blocking the interaction of ribosomes with mRNA by steric hindrance. See, e.g., Wittrup et al. Nat Rev Genet. (2015). 16(9):543-552.
  • an inhibitor is a short hairpin RNA.
  • a short hairpin RNA also known as a small hairpin RNA, is a single-stranded RNA that contains a hairpin, or loop, structure of unpaired bases. The hairpin is formed when a sequence in the RNA hybridizes with another sequence in the same RNA molecule through Watson-Crick base pairing, with the hairpin comprising the unpaired bases between the two complementary sequence.
  • shRNAs can be cleaved by the enzyme Dicer, resulting in the formation of double- stranded siRNAs that can inhibit gene expression as described previously. See, e.g., Moore et al. Methods Mol Biol. (2010). 629:141-158.
  • an inhibitor is a microRNA.
  • a microRNA is a small RNA molecule that can function in RNA interference and gene regulation.
  • a miRNA is generated from a longer RNA precursor known as primary miRNA (pri-miRNA), which is cleaved by the enzyme Drosha to form a shorter precursor miRNA (pre-miRNA) containing a hairpin.
  • pri-miRNA primary miRNA
  • pre-miRNA shorter precursor miRNA
  • Dicer Dicer to form a double- stranded intermediate RNA similar to a double- stranded siRNA, which can be integrated into a RISC and inhibit the expression of mRNAs containing a target sequence as described previously. See, e.g., O’Brien et al. Front Endocrinol. (2016). 9:402.
  • an inhibitor is a programmable nuclease.
  • a programmable nuclease is a protein that can be directed to cleave nucleic acids at a target site, such as a specific nucleotide sequence.
  • Programmable nucleases cut at or near target sequences, forming DNA double-stranded breaks. Cutting at a target sequence means cutting within the nucleotide sequence that is recognized by the programmable nuclease.
  • Cutting near a target sequence may be within 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides.
  • the programmable nuclease is a zinc-finger nuclease.
  • a zinc- finger nuclease (ZFN) is an endonuclease that can be programmed to cut specific sequences of DNA.
  • ZFNs are composed of a zinc-finger DNA-binding domain and a nuclease domain.
  • the DNA-binding domains of individual ZFNs generally contain 3-6 individual zinc finger repeats that recognize 9-18 nucleotides. For example, if the zinc finger domain perfectly recognizes a three base pair sequence, then a three-zinc finger array can be generated to recognize a nine-base pair target DNA sequence.
  • ZFNs with 4, 5, or 6 zinc finger domains are typically used to minimize off-target DNA cutting.
  • Non-limiting examples of zinc finger DNA-binding domains that may be used with methods of the present disclosure include Zif268, Gal4, HIV nucleocapsid protein, MYST family histone acetyltransferases, myelin transcription factor Myt1, and suppressor of tumorigenicity protein 18 (ST 18).
  • a ZFN may contain homogeneous DNA binding domains (all from the same source molecule) or a ZFN may contain heterogeneous DNA binding domains (at least one DNA binding domain is from a different source molecule).
  • Zinc finger DNA-binding domains work in concert with a nuclease domain to form a zinc finger nucleases (ZFNs) that cut target DNA (e.g., breakpoint junction).
  • ZFNs zinc finger nucleases
  • the nuclease cuts the DNA in a non-sequence specific manner after being recruited to the target DNA (e.g., breakpoint junction) by the zinc fingers DNA-binding domains.
  • the most widely-used ZFN is the type 2 restriction enzyme FokI, which forms a heterodimer before producing a double- stranded break in the DNA.
  • ZFNs may be nickases that only cleave one strand of the double-stranded DNA. By cleaving only one strand, the DNA is more likely to be repaired by error-free HR as opposed to error-prone NHEJ (see, e.g., Ramirez, et al. Nucleic Acids Research, 40(7): 5560-5568).
  • nucleases that may be used with methods in this disclosure include FokI and DNasel.
  • the programmable nuclease is a transcription activator-like effector nuclease.
  • a transcription activator-like effector nuclease (TALEN) is an endonuclease that can be programmed to cut specific sequences of DNA.
  • TALENs are composed of transcription activator-like effector (TALE) DNA-binding domains, which recognize single target nucleotides in the TNA, and transcription activator-like effector nucleases (TALENs) which cut the DNA at or near the target nucleotide.
  • TALE transcription activator-like effector
  • Transcription activator-like effectors (TALEs) found in bacteria are modular DNA binding domains that include central repeat domains made up of repetitive sequences of residues (see, e.g., Boch J.
  • the central repeat domains contain multiple repeat regions, with certain amino acids of the repeat region, known as the repeat variable diresidue (RVD) determining the nucleotide specificity of the TALE (see, e.g., Moscou MJ et al. Science 2009; 326 (5959): 1501; Juillerat A et al. Scientific Reports 2015; 5: 8150).
  • RVD repeat variable diresidue
  • TALE-based sequence detectors can recognize single nucleotides. Combining multiple repeat regions may produce sequence-specific synthetic TALEs (see, e.g., Cermak T et al. Nucleic Acids Research 2011; 39 (12): e82).
  • TALEs include IL2RG, AvrBs, dHax3, and thXoI.
  • the programmable nuclease is an RNA-guided nuclease.
  • RNA- guided nucleases are directed to a target sequence through the use of a guide RNA (gRNA) that is complementary to the target sequence.
  • gRNA guide RNA
  • a specific guide RNA may be utilized to direct the activity of the RNA-guided nuclease such that only the target sequence is cleaved.
  • the RNA-guided nuclease is a Clustered Regularly Interspace Palindromic Repeats-Associated (CRISPR/Cas) nuclease.
  • CRISPR/Cas nucleases exist in a variety of bacterial species, where they recognize and cut specific DNA sequences.
  • the CRISPR/Cas nuclease are grouped into two classes. Class 1 systems use a complex of multiple CRISPR/Cas proteins to bind and degrade nucleic acids, whereas Class 2 systems use a large, single protein for the same purpose.
  • a CRISPR/Cas nuclease as used herein may be selected from Cas9, CaslO, Cas3, Cas4, C2cl, C2c3, Casl3a, Casl3b, Casl3c, and Casl4 (see, e.g., Harrington, L.B. et al., Science, 2018 (DOI: 10.1126/scienceaav4294)).
  • Non-limiting examples of bacterial CRISPR/Cas9 nucleases for use herein include Streptococcus thermophilus Cas9, Streptococcus thermophilus CaslO, Streptooccus thermophilus Cas3, Staphylococcus aureus Cas9, Staphylococcus aureus CaslO, Staphylococcus aureus Cas3, Neisseria meningitidis Cas9, Neisseria meningitidis CaslO, Neisseria meningitidis Cas3, Streptococcus pyogenes Cas9, Streptococcus pyogenes CaslO, and Streptococcus pyogenes Cas3.
  • Other variant endonucleases may be used.
  • an RNA-guided nuclease is a CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1).
  • Cpf1 is a bacterial endonuclease similar to Cas9 nuclease in terms of activity. However, Cpf1 only requires a short ( ⁇ 42 nucleotide) gRNA, while Cas9 requires a longer (-100 nucleotide) gRNA. Additionally, Cpf1 cuts the DNA 5’ to the target sequence and leaves blunted ends, while Cas9 leaves sticky ends with DNA overhangs.
  • Cpf1 proteins from Acidaminococcus and Lachnospiraceae bacteria efficiently cut DNA in human cells in vitro.
  • the RNA-guided nuclease is Acidaminococcus Cpf1 or Lachnospiraceae Cpf1, which require shorter gRNAs than Cas nuclease proteins.
  • inhibitors such as antisense oligonucleotides (ASOs), RNAi molecules (e.g., shRNA, siRNA, or miRNA), and/or programmable nuclease-based gene editing molecules (e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs) are conditionally expressed in myofibers and/or osteoblasts.
  • ASOs antisense oligonucleotides
  • RNAi molecules e.g., shRNA, siRNA, or miRNA
  • programmable nuclease-based gene editing molecules e.g., CRISPR/Cas9/gRNAs, TALE/TALENs, and ZFNs
  • Expression may be quantified by measuring mRNA transcription, mRNA abundance, protein translation, and/or protein abundance using methods that are known in the art (e.g., qRT-PCR, Western blotting, immunoassays).
  • the frequency of expression in one environment relative to another may be evaluated by measuring expression in both environments and comparing the measurements using statistical methods that are known in the art (e.g., Student’s t-test, ANOVA, regression models).
  • An agent is said to have significantly more expression in a given environment relative to another environment if 1) the magnitude of expression as measured is higher in the given environment than in another environment, and 2) a statistical test or model determines that the probability of the results being due to chance is less than 0.1, less than 0.05, or less than 0.01.
  • This may be achieved by operably linking the gene to be expressed to a promoter that is active in the desired environment, but not other environments.
  • a promoter is said to be operably linked to a gene if the promoter controls the degree to which the gene is expressed.
  • Conditional expression of a gene in myofibers , for example, may be achieved through the use of the promoter.
  • promoters active in muscle cells include the promoter regions of Tnls, TnCf, and skAct. See, e.g., Corin et al. Proc Natl Acad Sci U SA. (1995). 92(13): 6185-6189.
  • Conditional expression of a gene in osteobalsts for example, may be achieved using the promoter region of any gene that is specifically active in osteoblasts.
  • Non- limiting examples of promoters active in osteoblasts include the promoter regions of the COL1A2 and OCN genes.
  • Type I receptors and type 2 receptors are receptors that associate with (e.g., bind to) activin or activin-like ligands.
  • Non-limiting examples of activin-like ligands include myostatin, TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3, inhibin a, inhibin pA, inhibin pB, inhibin pC, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, BMP15, GDF-1, GDF-2, GDF-3, GDF-4, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, GDF-13, GDF-14, GDF-15, nodal, and anti-mullerian hormone (AMH).
  • AMH anti-mullerian hormone
  • type 1 receptors and/or type 2 receptors Signaling through type 1 receptors and/or type 2 receptors occurs when activin, or an activin-like ligand such as myostatin, binds to a type 2 receptor, such as ACVR2A or ACVR2B, and the type 2 receptor then forms a complex with a type 1 receptor, such as ALK4 or ALK5.
  • This complex then recruits and phosphorylates proteins such as SMAD2 or SMAD3.
  • This SMAD2-SMAD3-SMAD4 complex then translocates to the nucleus, where it regulates gene expression.
  • Signal transduction may also include the inhibition of Akt, a serine/threonine kinase.
  • Akt Akt phosphorylates Forkhead transcription factors (FoxO proteins), and thus inhibition of Akt activity results in the generation of dephosphorylated FoxO as phosphate groups are lost from phosphorylated FoxO and not replaced.
  • Dephosphorylated FoxO translocates to the nucleus and activate transcription of E3 ubiquitin ligases MuRFl and Atrogin 1.
  • E3 ubiquitin ligases mark muscle contractile proteins for degradation by the proteasome, and so inhibition of Akt by myostatin, type 1 receptor(s), and/or type 2 receptor(s) can result in a reduction in muscle weight. See, e.g., Han et al. Curr Opin Support Palliat Care. (2011). 5(4):334— 341.
  • E3 ubiquitin ligases similarly mark proteins such as the osteocalcin required for bone formation, resulting in proteasomal degradation, decreased bone deposition, and reduced bone mineral density. See, e.g., Xi et al. J Recept Signal Transduct Res. 2015. 35(6): 640-645.
  • Type I receptors are required for signaling by activin and other activin-like ligands such as myostatin. Following association between a ligand and a type 2 receptor, the type 2 receptor associates with a type 1 receptor, after which signal transduction occurs.
  • the data provided herein show that type 1 receptors are functionally redundant with each other, such that the deletion of one type 1 receptor is insufficient to abrogate myostatin and/or activin signaling in a subject, but the deletion of multiple type 1 receptors reduces myostatin and/or activin signaling and thus increases muscle weight, improves blood glucose, reduces body fat content, and increases bone density in a subject.
  • the type 1 receptor is ALK4, also referred to as activin receptor type- IB (ACVR1B).
  • ALK4 is encoded by the ACVR1B gene, and acts as a transducer of signals activin or activin-like ligands.
  • Non-limiting examples of ligands that interact with ALK4 are myostatin, activin A, activin B, activin AB, nodal, GDF-1, GDF-3, GDF-8, GDF-11, BMP11, TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3 and Vg1.
  • ALK4 inhibitors i.e.
  • agents that inhibit signaling through ALK4 include follistatin, inhibin A, inhibin B, left-right determination factor 1, left-right determination factor 2, A 83-01, SB-431542, SB-505124, EW-7197, K02288, LDN-212854, LY-364947, LY-2157299, Galunisertib, GW-6604, SD-208, AZ12799734, Vactosertib, EW-7195, TP-008, E616452, SB525334, SJN2511, AZ12601011, GW 788388, and SM16. See, e.g., Cui et al. Mol Med Rep. (2019).
  • amino acid sequence for human ALK4 is given by Accession No. P36896-1 and is reproduced as SEQ ID NO: 2.
  • the type 1 receptor is ALK5, also referred to as transforming growth factor P receptor I or TGF ⁇ RI.
  • ALK5 is encoded by the TGFBR1 gene, and acts as a receptor for activins, which belong the TGF ⁇ superfamily of signaling ligands.
  • Non-limiting examples of ligands that interact with ALK5 are myostatin, avotermin, GDF-10, BMP3B, GDF- 11, BMP11, TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3.
  • ALK5 inhibitors i.e.
  • agents that inhibit signaling through ALK5 include follistatin, fresolimumab, lerdelimumab, metelimumab, A 83-01, D-4476, GW-788388, LY-364947, LY-2109761, Galunisertib (LY- 2157299), R-268712, RepSox (E-616452, SJN-2511), SB-431542, SB-505124, SB-525334, SD- 208, EW-7197, K02288, LDN-212854, GW-6604, AZ12799734, Vactosertib, EW-7195, TP- 008, AZ12601011, and SM16.
  • amino acid sequence for human ALK5 is given by Accession No. P36897 and is reproduced as SEQ ID NO: 4.
  • Some aspects of the present disclosure provide methods of administering inhibitors of type 2 receptors signaling to a subject.
  • a type 2 receptor after association with activin or an activin-like ligand such as myostatin, can dimerize with a type 1 receptor, resulting in signal transduction.
  • the data provided herein show that type 2 receptors are functionally redundant with each other, such that the deletion of one type 2 receptor is insufficient to abrogate myostatin signaling in a subject, but the deletion of multiple type 2 receptors reduces myostatin signaling and thus increases muscle weight, improves blood glucose, reduces body fat content, and increases bone density in a subject.
  • the type 2 receptor is ACVR2A, also referred to as activin receptor type-2A or ACVR2.
  • ACVR2A is encoded by the ACVR2A gene, and acts as a receptor for activins, which belong the TGF ⁇ superfamily of signaling ligands.
  • Non-limiting examples of ligands that interact with ACVR2A are myostatin, activin A, activin B, activin AB, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP11, BMP12, BMP13, BMP14, BMP15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-9, GDF-8, GDF-11, GDF-15, dibotermin alfa, eptotermin alfa, nodal, and radotermin.
  • ACVR2A inhibitors i.e.
  • agents that inhibit signaling through ACVR2A include follistatin, inhibin A, inhibin B, left- right determination factor 1, left-right determination factor 2, sotarercept, CZC24758, dorsomorphin, LDN-193189, bimagrumab, CDD861, BYM338, ACVR2A/Fc, and ACVR2- ECD. See, e.g., Goh et al. J Biol Chem (2017). 292(33): 13809-13822.
  • ACVR2A An example of an amino acid sequence of human ACVR2A is given by Accession No. P27037 and is reproduced as SEQ ID NO: 6.
  • the type 2 receptor is ACVR2B, also referred to as activin receptor type-2B.
  • ACVR2B is encoded by the ACVR2B gene, and acts as a receptor for activins, which belong the TGF ⁇ superfamily of signaling ligands.
  • Non-limiting examples of ligands that interact with ACVR2B are myostatin, activin A, activin B, activin AB, BMP1, BMP2, BMP3, BMP3A, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP11, BMP12, BMP13, BMP14, BMP15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-9, GDF-8, GDF-11, GDF-15, dibotermin alfa, eptotermin alfa, nodal, and osteogenin, and radotermin.
  • Non-limiting examples of ACVR2B inhibitors include follistatin, inhibin A, inhibin B, left-right determination factor 1, left-right determination factor 2, ramatercept, CZC24758, dorsomorphin, LDN-193189, bimagrumab, CDD861, BYM338, DLK1, RAP-031, ACVR2B/Fc, and ACVR2B-ECD. See, e.g., Formicola et al. Front Physiol. (2016). 9:515; Sako et al. J Biol Chem. (2010). 285(27): 21037-21048; Goh et al. J Biol Chem. (2017) 292(33): 13809-13822.
  • the type 2 receptor is transforming growth factor ⁇ receptor II (TGF ⁇ RII).
  • TGF ⁇ RII is encoded by the TGFBR2 gene, and acts as a receptor forTGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3, which belong the TGF ⁇ superfamily of signaling ligands.
  • TGF ⁇ RII inhibitors e.g., agents that inhibit signaling through TGF ⁇ RII
  • agents that inhibit signaling through TGF ⁇ RII include follistatin, fresolimumab, lerdelimumab, metelimumab, ITD-1 DMH-2, LY-364947, LY- 2109761, galunisertib (LY-2157299), compound 13a (PMID: 23639540), compound 13d (PMID: 23639540), and compound 15b (PMID: 16539403).
  • follistatin e.g., fresolimumab, lerdelimumab, metelimumab, ITD-1 DMH-2, LY-364947, LY- 2109761, galunisertib (LY-2157299), compound 13a (PMID: 23639540), compound 13d (PMID: 23639540), and compound 15b (PMID: 16539403).
  • TGF ⁇ transforming growth factor beta
  • TGF ⁇ 1 regulates the proliferation, differentiation, and activation of multiple cell types, including T cells, B cells, myeloid cells, muscle cells, osteoblasts, and osteoclasts.
  • TGF ⁇ 2 modulates multiple processes such as cellular metabolism, embryonic development, and tumor suppression.
  • TGF ⁇ 3 regulates cellular adhesion, mammalian development, and wound healing.
  • TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3 interact with ALK5 and TGF ⁇ RII.
  • Inhibition of TGF ⁇ RII signaling may thus be achieved through the use of an agent or combination of agents that bind(s) to TGF ⁇ RII or the use of an agent or combination of agents that bind(s) to TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3.
  • inhibition of TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3 signaling may be achieved through the use of an agent or combination of agents that bind(s) to TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3, or the use of an agent or combination of agents that bind(s) to TGF ⁇ RII.
  • the TGF ⁇ superfamily also includes myostatin and activin A, which act as negative regulators of skeletal muscle growth, osteoblast differentiation, and bone deposition.
  • the data provided herein show that inhibiting myostatin signaling and/or signaling through type 1 and/or type 2 receptors, either through targeted inhibition of myostatin, or receptors that associate with myostatin and other TGF ⁇ cytokines, results in increased muscle mass and bone mineral density in a subject. See, e.g., Hata et al. Cold Spring Harb Perspect Biol. (2016). 8(9):a022061.
  • compositions comprising an agent or combination of agents that inhibit signaling through type 1 receptors and/or type 2 receptors.
  • the composition comprises an agent or combination of agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII.
  • agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII are co-formulated (present in the same composition).
  • the composition is administered in an effective amount.
  • an effective amount refers to the amount (e.g., dose) at which a desired clinical result (e.g., muscle growth and/or bone deposition) is achieved in a subject.
  • An effective amount is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the inhibitor, other components of the composition, and other determinants, such as age, body weight, height, sex and general health of the subject.
  • a subject may be a mammal, such as a human, a non-human primate (e.g., Rhesus monkey, chimpanzee), or a rodent (e.g., a mouse or a rat). In some embodiments, the subject is a human subject.
  • the subject has a disease associated with myostatin signaling. In some embodiments, the subject has a disease associated with type 1 and/or type 2 receptor signaling. In some embodiments, the subject has a disease associated with type 1 and/or type 2 receptor signaling in muscle cells. In some embodiments, the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, muscle atrophy, spinal muscle atrophy, age-related sarcopenia, Charcot-Marie-Tooth disease, cachexia, chronic obstructive pulmonary disease, kidney disease, or cancer. In some embodiments, the subject has a disease associated with type 1 and/or type 2 receptor signaling in osteoblasts. In some embodiments, the subject has osteoporosis, Cushing’s syndrome, pituitary disorders, and hyperthyroidism.
  • DMD Duchenne muscular dystrophy
  • DMD is a genetic disorder characterized by progressive muscle degeneration and weakness due to a mutation in the DMD gene, which encodes the dystrophin protein, a critical protein for muscle cell function.
  • FSHD facioscapulohumeral muscular dystrophy
  • compositions and methods of use for treating inclusion body myositis IBM is an inflammatory muscle disease of unknown cause characterized by progressive muscle degeneration due to the infiltration of muscle tissue by immune cells, deposition of abnormal proteins, and filamentous inclusions in muscle fibers.
  • Muscle atrophy is the loss of skeletal muscle mass due to one of many potential causes, including disuse, immobility, aging, malnutrition, or injury.
  • SMA spinal muscular atrophy
  • Age-related sarcopenia is the degenerative loss of muscle mass and strength that occurs with aging, involving reduction in the number of muscle fibers and loss of muscle regeneration activity.
  • CMT Charcot-Marie-Tooth disease
  • CMT is a genetic disorder of the nervous system characterized by the progressive loss of muscle tissue in multiple parts of the body.
  • Some aspects of the present disclosure provide compositions and methods of use for treating cachexia.
  • Cachexia is a complex syndrome resulting in loss of muscle tissue due to increased proteolysis, decreased protein synthesis, and signaling of TGF ⁇ and activin in muscle cells.
  • Cachexia is often associated with chronic obstructive pulmonary disease (an inflammatory disease that blocks airflow to and from the lungs, impairing breathing), kidney disease (disruption in normal kidney function), and cancer (unregulated cell growth resulting in tumor formation and/or disruption of healthy physiology in the affected organ(s)).
  • Osteoporosis is skeletal disorder characterized by low bone mineral density and deterioration of bone tissue. Individuals with osteoporosis experience fragility in deteriorating bones, and are at increased risk for fractures, particularly in vertebrae, arms, and hips.
  • Cushing’s syndrome is a condition resulting from prolonged exposure to glucocorticoid hormones, such as cortisol. Multiple signs and symptoms are associated with Cushing’s syndrome, including weak bones, which present an increased risk for fractures.
  • compositions and methods of use for treating a pituitary disorder are associated with abnormal activity of the pituitary gland, and consequently abnormal development. Hormone imbalances associated with pituitary disorders can hinder the accumulation of bone mineral density during development and puberty, and thus place individuals with pituitary disorders at greater risk for bone weakness and fractures.
  • Hyperthyroidism is a condition characterized by overproduction of the thyroid hormone thyroxine. Hyperthyroidism is associated with accelerated bone remodeling by osteoclasts, leading to reduced bone density, osteoporosis, and increased risk of fractures.
  • a composition is a pharmaceutical composition.
  • a pharmaceutical composition is a combination of an (at least one) active agent, such as an ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor, with an excipient, inert or active, making the composition especially suitable for therapeutic use in vivo or ex vivo.
  • a pharmaceutically acceptable excipient can also be incorporated in a formulation and can be any excipient (e.g., carrier) known in the art.
  • Non-limiting examples include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based mixtures and emulsion-based mixtures of such carriers.
  • any pharmaceutically acceptable excipients are known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters
  • each pharmaceutically acceptable excipients used in a pharmaceutical composition of the invention must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • Excipients suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
  • excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with DNA or RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
  • Relative amounts of the active agent, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1- 30%, between 5-80%, or at least 80% (w/w) active ingredient.
  • the ratio of a first inhibitor to a second inhibitor in a composition may vary.
  • the ratio of the first inhibitor to the second inhibitor is 1:1 to 1:10, or 1:1 to 1:5.
  • the ratio of ALK4 inhibitor to ALK5 inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8., 1:9, or 1:10.
  • the ratio of a second inhibitor to a first inhibitor is 1:1 to 1:10, or 1:1 to 1:5.
  • the ratio of ALK5 inhibitor to ALK4 inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the ratio of a first inhibitor to a second inhibitor in a composition may vary. In some embodiments, the ratio of the first inhibitor to the second inhibitor is 1:1 to 1:10, or 1:1 to 1:5. For example, the ratio of ACVR2A inhibitor to ALK5 inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8., 1:9, or 1:10. In other embodiments, the ratio of a second inhibitor to a first inhibitor is 1:1 to 1:10, or 1:1 to 1:5. For example, the ratio of ALK5 inhibitor to ACVR2A inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the ratio of a first inhibitor to a second inhibitor in a composition may vary. In some embodiments, the ratio of the first inhibitor to the second inhibitor is 1:1 to 1:10, or 1:1 to 1:5.
  • the ratio of ACVR2B inhibitor to ALK5 inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8., 1:9, or 1:10.
  • the ratio of a second inhibitor to a first inhibitor is 1:1 to 1:10, or 1:1 to 1:5.
  • the ratio of ALK5 inhibitor to ACVR2B inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the ratio of a first inhibitor to a second inhibitor in a composition may vary.
  • the ratio of the first inhibitor to the second inhibitor is 1:1 to 1:10, or 1:1 to 1:5.
  • the ratio of ACVR2A inhibitor to ACVR2B inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8., 1:9, or 1:10.
  • the ratio of a second inhibitor to a first inhibitor is 1:1 to 1:10, or 1:1 to 1:5.
  • the ratio of ACVR2B inhibitor to ACVR2A inhibitor may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the composition comprises an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4, and ALK5. In some embodiments, the composition comprises an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4, and ACVR2A. In some embodiments, the composition comprises an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4, and ACVR2B. In some embodiments, the composition comprises an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5, and ACVR2A. In some embodiments, the composition comprises an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5, and ACVR2B. In some embodiments, the composition comprises an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ACVR2A, and ACVR2B.
  • Some aspects of the present disclosure provide methods for treating diseases associated with myostatin signaling by administering to a subject an agent or combination of agents that inhibit signaling through type 1 receptors and/or type 2 receptors.
  • the method comprises administering to a subject (e.g., a human subject having muscular dystrophy) a composition comprising an agent or combination of agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII.
  • routes of administration include oral (e.g., tablet, capsule), intravenous, intramuscular, intraperitoneal, subcutaneous, intranasal, and intratumoral.
  • At least two inhibitors are administered sequentially.
  • an ALK4 inhibitor may be administered before or after (e.g., on the order of minutes, hours, or days before or after) an ALK5 inhibitor.
  • at least two inhibitors are administered concomitantly (at the same time).
  • an ALK4 inhibitor and an ALK5 inhibitor may be formulated in the same composition.
  • At least two inhibitors are administered sequentially.
  • an ACVR2A inhibitor may be administered before or after (e.g., on the order of minutes, hours, or days before or after) an ALK5 inhibitor.
  • an ACVR2B inhibitor may be administered before or after (e.g., on the order of minutes, hours, or days before or after) an ALK5 inhibitor.
  • an ACVR2A inhibitor may be administered before or after (e.g., on the order of minutes, hours, or days before or after) an ACVR2B inhibitor.
  • At least two inhibitors are administered concomitantly (at the same time).
  • an ACVR2A inhibitor and an ALK5 inhibitor may be formulated in the same composition.
  • an ACVR2B inhibitor and an ALK5 inhibitor may be formulated in the same composition.
  • an ACVR2A inhibitor and an ACVR2B inhibitor may be formulated in the same composition.
  • the method comprises administering to the subject an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4, and/or ALK5. In some embodiments, the method comprises administering to the subject an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4, and/or ACVR2A. In some embodiments, the method comprises administering to the subject an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK4, and/or ACVR2B. In some embodiments, the method comprises administering to the subject an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5, and/or ACVR2A.
  • the method comprises administering to the subject an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ALK5, and/or ACVR2B. In some embodiments, the method comprises administering to the subject an agent or combination of agents that inhibit(s) signaling through TGF ⁇ RII, ACVR2A, and ACVR2B. In some embodiments, the dose of ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor administered to a subject is equivalent to (e.g., within 10% of), or lower than (e.g., at least 0.5-fold, at least 1-fold, at least 2-fold lower than), a control standard-of-care dose.
  • a standard-of-care refers to a medical treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance.
  • a standard-of-care dose as provided herein refers to the dose of ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor that a physician/clinician or other medical professional would administer to a subject to treat or prevent cancer, while following the standard of care guideline for treating or preventing a disease associated with myostatin signaling.
  • the dose of the inhibitor administered to a subject is a standard- of-care dose. In some embodiments, the dose of the inhibitor administered to a subject is at least 10% lower than the standard-of-care dose for the inhibitor. For example, the dose of ALK4 inhibitor administered to a subject is at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% less than the standard-of-care dose for the inhibitor. In some embodiments, the dose of inhibitor administered to a subject is 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, or 40%-50% less than the standard-of-care dose for the inhibitor.
  • the ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor are administered in an amount effective to increase muscle weight in the subject relative to a control (e.g., baseline, prior to administration of the inhibitors, or following administration of only one of the inhibitors).
  • the control may be a subject that is not administered a composition, or is administered a composition that does not contain any agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII (e.g. a composition containing only a pharmaceutically acceptable excipient).
  • the baseline may be the muscle weight of a subject as measured before administration of a composition.
  • Muscle weight also known as muscle mass, is the total weight or mass of muscle present in a body, organ, or particular muscle. Muscle weight may include the weight of skeletal muscle, smooth muscle, and cardiac muscle. Muscle weight may be quantified by measuring lean body mass (total body weight minus body fat weight), muscle volume (total size of muscle), and/or muscle cross-sectional area (area of the cross section of a muscle, generally at its largest point), and calculating muscle weight accordingly. Muscle weight, lean body mass, muscle volume, and muscle cross-sectional area in a subject may be determined and calculated using any number of muscle measurement methods that are known in the art and apparent to one of ordinary skill in the art (e.g..).
  • muscle weight is increased by at least 3%, at least 4%, at least 5%, least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, or at least 150% relative to a control or baseline.
  • muscle weight is increased by 3-300%, 3-250%, 3-100%, 3-80%, 3-50%, 3-25%, 3-10%, 3-5%, 5-300%, 5- 250%, 5-100%, 5-80%, or 5-50%, 10-300%, 10-250%, 10-100%, 10-80%, or 10-50%, 25- 300%, 25-250%, 25-100%, 25-80%, or 25-50% relative to a control or baseline.
  • Non-limiting examples of muscles in which weight may be increased include the tricep, quadricep, gastrocnemius, plantaris, pectoralis, trapezius, latissimus, romboid, levator scapulae, subclavius, serratus, deltoid, teres, supraspinatus, infraspinatus, subscapularis, brachialis, anconeus, pronator, radialis, palmaris, ulnaris, pronator, flexor digitorum, flexor pollicis, extensor digitorum, extensor digitii, brachioradialis, supinator, extensor indicis, opponens, abductor, adductor, and tibialis muscles.
  • the ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor are administered in an amount effective to increase bone weight in the subject relative to a control (e.g., baseline, prior to administration of the inhibitors, or following administration of only one of the inhibitors).
  • the control may be a subject that is not administered a composition, or is administered a composition that does not contain any agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII (e.g. a composition containing only a pharmaceutically acceptable excipient).
  • the baseline may be the muscle weight of a subject as measured before administration of a composition.
  • Bone weight also known as bone mineral density, is the total weight or mass of bone present in a body or particular bone.
  • Muscle weight may include the weight of skeletal muscle, smooth muscle, and cardiac muscle. Bone weight and density in a subject may be determined and calculated using any number of muscle measurement methods that are known in the art and apparent to one of ordinary skill in the art (e.g., dual X-ray absorptiometry, quantitative CT scanning, and ultrasonography) (Sheu and Diamond. Aust Prescr. 2016. 39(2):35— 39).).
  • bone weight is increased by at least 3%, at least 4%, at least 5%, least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, or at least 150% relative to a control or baseline.
  • bone weight is increased by 3-300%, 3-250%, 3-100%, 3-80%, 3-50%, 3-25%, 3-10%, 3-5%, 5- 300%, 5-250%, 5-100%, 5-80%, or 5-50%, 10-300%, 10-250%, 10-100%, 10-80%, or 10- 50%, 25-300%, 25-250%, 25-100%, 25-80%, or 25-50% relative to a control or baseline.
  • Non- limiting examples of bones in which bone mineral density may be increased include the occipital bone, parietal bone, frontal bone, temporal bone, sphenoid bone, ethmoid bone, mandible, humerus, scapula, clavicle, ulna, radius, carpals metacarpals, phalanges, cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacrum, coccyx, pelvis, femur, patella, tibia, fibula, tarsals, and metatarsals.
  • the ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor are administered in an amount effective to improve glucose metabolism in the subject relative to a control (e.g.. baseline, prior to administration of the inhibitors, or following administration of only one of the inhibitors).
  • the control may be a subject that is not administered a composition, or is administered a composition that does not contain any agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII (e.g. a composition containing only a pharmaceutically acceptable excipient).
  • the baseline may be the glucose metabolism of a subject as measured before administration of a composition.
  • Glucose metabolism refers to the process of breaking down glucose and/or converting glucose to other molecules, as well as the rate at which these processes occur. Glucose metabolism in a subject may be measured using any number of methods that are known in the art and apparent to one of ordinary skill in the art (e.g., blood glucose concentration, insulin secretion, and magnetic resonance imaging) (Ayala et al. Dis Model Meeh. (2010). 3(9— 10):525— 534).
  • glucose metabolism is improved by at least 5%, least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, or at least 150% relative to a control or baseline.
  • glucose metabolism is improved by 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 10-75%, 20-75%, 30-100%, 40-75%, 50-75%, 10-50%, 20-50%, 30-50%, or 40-50%, relative to a control or baseline.
  • Glucose metabolism may also be defined as the magnitude of reduction in blood glucose in a given amount of time. In some embodiments, at least 5%, least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, or at least 150% more glucose is metabolized in a given time, relative to a control or baseline.
  • the ALK4 inhibitor, ALK5 inhibitor, ACVR2A inhibitor, ACVR2B inhibitor, and/or TGF ⁇ RII inhibitor are administered in an amount effective to reduce body fat content in the subject relative to a control (e.g.. baseline, prior to administration of the inhibitors, or following administration of only one of the inhibitors).
  • the control may be a subject that is not administered a composition, or is administered a composition that does not contain any agents that inhibit signaling through ALK4, ALK5, ACVR2A, ACVR2B, and/or TGF ⁇ RII (e.g. a composition containing only a pharmaceutically acceptable excipient).
  • the baseline may be the body fat content of a subject as measured before administration of a composition.
  • Body fat content also known as body fat percentage, is the total mass of fat contained in a body divided by the total mass of the body and expressed as a proportion or percentage.
  • Body fat content in a subject may be determined using any number of body fat measurement methods that are known in the art and apparent to one of ordinary skill in the art (e.g.. skin calipers, bioelectrical impedance, hydrostatic weighing, dual-energy X-ray absorptiometry, air-displacement plethysmography, 3-dimensional body scanning, and magnetic resonance imaging) (Lemos et al. Curr Opin Endocrinol Diabetes Obes. (2017). 24(5):310- 314).
  • body fat content is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% relative to a control or baseline.
  • body fat content is reduced by 5- 100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 10-75%, 20-75%, 30-100%, 40- 75%, 50-75%, 10-50%, 20-50%, 30-50%, or 40-50%, relative to a control or baseline.
  • Example 1 Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A
  • mice carrying floxed alleles for both Acvr2 and Acvr2b were generated and these alleles were targeted using a transgene expressing Cre recombinase from a myosin light chain promoter/enhancer (Myl1 -Cre), which is expressed by skeletal muscle fibers but not by satellite cells (23).
  • Myl1 -Cre myosin light chain promoter/enhancer
  • the Myl1 -Cre transgene significantly reduced RNA levels for both Alk4 and Alk5 in muscle (data not shown).
  • Targeting Alk4 or Alk5 in myofibers resulted in statistically significant effects on muscle mass, ranging from up to 11% in the case of Alk4 and up to 18% in the case of Alk5 depending on the sex of the mice and the specific muscles examined (FIG. 1B).
  • targeting both type I receptors simultaneously resulted in much more substantial increases, with the greatest effects being seen in the quadriceps (173% and 136% in females and males, respectively) and gastrocnemius (249% and 197% in females and males, respectively).
  • mice carrying a floxed TGF ⁇ RII allele were used to examine the possibility that MSTN and/or activin A may signal through BMPRII in muscle.
  • Mice in which TGF ⁇ RII was targeted in myofibers generally had lower muscle weights than ere negative mice, with the effects being more pronounced in females (7- 13% depending on the muscle).
  • Mice in which all three type II receptors ( TGF ⁇ RII, Acvr2, and Acvr2b) were targeted had muscle weights that were comparable to those seen in mice in which just Acvr2 and Acvr2b were targeted.
  • the effects of targeting Alk4 in combination with Alk5 were striking not only in terms of their magnitude but also in terms of the variability from mouse to mouse.
  • the weight of the gastrocnemius muscle was relatively consistent in wild type and Mstn -/- mice as well as in mice in which both Acvr2 and Acvr2b were targeted in myofibers.
  • the weight of the gastrocnemius muscle was highly variable in mice targeting both Alk4 and Alk5 in myofibers, ranging from wild type levels in some mice to over 5 times wild type levels in other mice.
  • Cripto Another receptor component that has been implicated in MSTN signaling is Cripto (Cfc1b), which is known to serve as a co-receptor for certain ligands and to antagonize the activity of other ligands.
  • Cfc1b Cripto
  • C2C12 myoblasts reported that Cripto is required for MSTN signaling but inhibits activin A signaling; another study, however, showed that during muscle regeneration in vivo, Cripto expressed by satellite cells acts to antagonize MSTN signaling.
  • the Myl1-Cre transgene was used to target Cfc1b either alone or in combination with each of the type I or type II receptors. As shown in FIG.
  • the rationale was that by simultaneously targeting, for example, both Mstn and one type II (or type I) receptor, the contribution of the other type II (or type I) receptor in mediating activin A signaling could be determined.
  • the injured muscles were examined at 5 days and 21 days post-injury (DPI) to assess the effect of myofiber- specific knockout of Acvr2 and Acvr2b on muscle regeneration.
  • DPI post-injury
  • fiber cross sectional area (CSA) was significantly greater in uninjured muscles lacking both Acvr2 and Acvr2b.
  • no differences in fiber CSA were observed in injured muscles assessed at 5 DPI and 21 DPI.
  • Mstn -/- mice exhibit a significant suppression of fat accumulation and improved glucose metabolism in an otherwise wild type background as well as in ob/ob and agouti lethal yellow backgrounds. Beneficial metabolic effects have also been described in mice treated with MSTN inhibitors. A key question is whether these beneficial effects on fat accumulation and glucose metabolism are the result of inhibition of MSTN signaling to myofibers, leading to muscle growth, or whether they reflect lack of direct MSTN signaling to other cell types and tissues, including adipose tissue. Previous studies showed that differences in fat accumulation between Mstn -/- and wild type C57BL/6 mice become more pronounced as mice age.
  • mice The effects of placing these mice on high fat diets were also observed. Younger mice (12 weeks of age) of both sexes were monitored in these studies. Although effects were generally similar in both males and females, they were more pronounced in males. As shown in FIG. 4A, Mstn -/- mice gained much less weight than wild type mice throughout an 8-week period on a high fat diet. Similarly, Acvr2 fl/fl-Acvr2b fl/fl mice carrying the Myl1 -Cre transgene gained significantly less weight on a high fat diet than mice lacking Cre. These mice also differed in terms of glucose metabolism when maintained on a high fat diet.
  • At least part of this effect is due to inhibition of signaling directly to bone (likely by activin A), as targeting Acvr2 and Acvr2b in osteoblasts is sufficient to increase bone density in vivo. It is also possible, however, that some of the effects on bone may be indirect as a result of increased mechanical load on the bone due to enhanced muscle mass resulting from inhibition of signaling to myofibers.
  • the bones of mice in which Acvr2 and Acvr2b were targeted in myofibers were compared to the bones of wild type mice, Cre-negative mice, and mice receiving the ACVR2B/Fc decoy receptor.
  • ACVR2B is the primary type II receptor utilized by MSTN in vivo.
  • mice null for either Acvr2 or Acvr2b exhibit increased muscling and that these two receptors are partially functionally redundant.
  • One limitation of that study was that examining the consequence of complete loss of both receptors was not possible using the deletion alleles for these genes because mice completely lacking both receptors are not viable.
  • these receptors were targeted specifically in myofibers, as myofibers are direct targets for MSTN signaling and that blocking signaling in myofibers is sufficient to induce muscle growth.
  • These receptors were targeted by crossing in a myosin light chain-Cre (Myl1 -Cre) transgene, which is expressed specifically by myofibers, into mice carrying floxed alleles for Acvr2 and Acvr2b.
  • Myl1 -Cre myosin light chain-Cre
  • Targeting Acvr2 and Acvr2b individually resulted in small, though significant, increases in muscle mass but that simultaneously targeting both type II receptors leads to much more dramatic effects, demonstrating conclusively that both receptors function to suppress muscle growth and that the two receptors are functionally redundant in this regard.
  • ACVR2 and ACVR2B as MSTN receptors led to two strategies to develop therapeutics targeting MSTN signaling to treat patients with muscle loss or degeneration.
  • One approach was to generate a soluble form of ACVR2B in which the ligand- binding domain was fused to an immunoglobulin Fc domain.
  • this decoy receptor ACVR2B/Fc is still the most potent agent described to date in terms of its ability to promote muscle growth; in fact, just two injections of this decoy receptor at high doses to mice can cause greater than 50% muscle growth throughout the body in just two weeks.
  • mice Although targeting the two type I receptors generated the greatest effects on muscle mass, the phenotype was highly variable in these mice, and further experiments therefore focused on mice in which the two type II receptors, ACVR2 and ACVR2B, were targeted, as the phenotype was much more consistent.
  • Mstn -I- mice One tissue other than skeletal muscle known to be affected in Mstn -I- mice is adipose tissue.
  • Mstn -I- mice have a reduction in fat accumulation, particularly as a function of age, not only in a wild type background but also in ob/ob and agouti lethal yellow backgrounds, as well as beneficial effects on glucose metabolism.
  • loss or inhibition of MSTN can increase skeletal muscle glucose uptake and energy expenditure and protect against high fat diet- induced weight gain as well as glucose intolerance.
  • a key question is whether each effect on adipose tissue and glucose metabolism reflects a loss of MSTN signaling to skeletal muscle, or some effects reflect a loss of MSTN signaling to other tissues.
  • mice overexpressing a truncated form of ACVR2B in skeletal muscle also exhibit some of the metabolic effects seen in Mstn -I- mice.
  • this truncated receptor could act as a ligand trap, one possibility is that one mode of action of this truncated receptor may be to act as a sink by binding MSTN produced by skeletal muscle and thereby leading to inhibition of MSTN signaling not only to muscle but also to other tissues.
  • adipocytes among the cell types known to be responsive to MSTN in cell culture are adipocytes.
  • Mstn is expressed at low levels in adipose tissue in wild type mice
  • Mstn expression is significantly upregulated in both subcutaneous and visceral fat in ob/ob mice.
  • the metabolic effects of targeting Acvr2 and Acvr2b in myofibers were analyzed. These mice, like Mstn -I- mice, had reduced overall body fat, lower serum leptin levels, and reduced weight gain on a high fat diet.
  • These receptor-targeted mice also had lower fasting blood glucose despite having lower fasting insulin levels and are able to maintain lower glucose levels in glucose tolerance tests.
  • Mstn -I- mice Another tissue known to be affected in Mstn -I- mice is bone.
  • Mstn -I- mice have been reported to have a generalized increase in bone mineral density at many sites, including femurs.
  • a key question raised by these findings is whether this increased bone mineral density results from increased mechanical load on the bones due to the hypermuscularity in these mice or rather from loss of MSTN signaling directly to bone.
  • MSTN has been reported to be capable to acting directly on bone progenitor cells in vitro to regulate cell differentiation.
  • MSTN inhibitors like follistatin and the ACVR2B/Fc decoy receptor, can have significant effects on bone repair and bone density in vivo, but because these inhibitors can also block activin signaling, the identities of the key ligands being blocked in these studies is not clear.
  • targeting type II receptors in osteoblasts in vivo can also increase bone density, but this effect likely reflects inhibition of signaling by activin rather than by MSTN.
  • the bones of mice in which Acvr2 and Acvr2b were in myofibers were analyzed.
  • ES cell colonies carrying the homologously- targeted allele were injected into blastocysts, and mice generated from these blastocysts were bred to identify those exhibiting germ-line transmission of the targeted allele.
  • Offspring from these matings were then bred with EII ⁇ -Cre transgenic mice in order to delete the neomycin resistance cassette in the germ-line. From these crosses, mice carrying an Alk4 flox allele lacking the NEO cassette were obtained.
  • TA muscles were dissected from both sides of 10-week-old mice, and the average weight was used for each muscle. Circulating MSTN levels were determined on acid activated serum samples by ELISA using the R&D Systems DGDF80 kit. To induce muscle damage and regeneration, 50 ⁇ L 1.2% barium chloride (w/v) was delivered to the right TA muscle over ten intramuscular punctures. The left TA served as the uninjured control. TA muscles were harvested either 5 days or 21 days post injury, mounted in OCT, and frozen in thawing isopentane. Serial sections (8 ⁇ m) were cut transversely through the belly of the TA muscle using a refrigerated cryostat.
  • TA sections were immunoreacted to laminin and Pax7 applied with the M.O.M Basic Kit. Sections were then counterstained with DAPI to visualize nuclei and imaged with a Zeiss Observer Z1 microscope with a color camera controlled by Volocity software. Images were then quantified using ImageJ software.
  • Live animal imaging was performed using a Piximus dual-energy X-ray absorptiometer (DXA).
  • DXA Piximus dual-energy X-ray absorptiometer
  • GTT Glucose tolerance tests
  • Mice were then placed on a 60 kcal% fat diet (D 12492) for 8 weeks, with a repeat GTT being performed after 4 weeks.
  • the ACVR2B/Fc decoy receptor was expressed in Chinese hamster ovary cells, purified from the conditioned medium using a protein A Sepharose column, and administered intraperitoneally at a dose of 175 pg per injection.
  • the left femur and lumbar vertebrae were placed in 70% ethanol.
  • ⁇ CT was performed in a Scanco ⁇ CT40 at 8pm3 resolution. Samples were scanned in 70% ethanol 55kVp, 145 ⁇ A intensity, 300ms. The instrument is calibrated weekly using Scanco phantoms, and all scans passed routine quality control verification. Analysis of femurs and vertebrae was conducted using standard protocols, with a lower threshold of 2485 Hounsfield units (HU) for femoral trabeculae, 4932HU for femoral cortex, and 3078HU for vertebral trabeculae. Surface renderings were generated corresponding to each of these thresholds.
  • Example 2 Local versus systemic control of bone and skeletal muscle mass by components of the transforming growth factor- ⁇ signaling pathway
  • MSTN Myostatin
  • TGF- ⁇ transforming growth factor- ⁇
  • Mice lacking MSTN exhibit dramatic increases in skeletal muscle mass throughout the body, with individual muscles growing to about twice the normal size.
  • the amino acid sequence of MSTN has been strongly conserved through evolution (2) and engineered or naturally-occurring mutations in the MSTN gene have been shown to lead to increased muscling in many other species as well, including cattle (2-4), sheep (5), dogs (6), rabbits (7), rats (8), swine (9), goats (10), and humans (11).
  • MSTN activity is regulated by various extracellular binding proteins, including follistatin (FST) (12), FSTL-3 (13), GASP-1 (14), and GASP-2 (15, 16) as well as the MSTN propeptide, which maintains MSTN in an inactive, latent state (12, 17-19).
  • FST follistatin
  • MSTN propeptide which maintains MSTN in an inactive, latent state (12, 17-19).
  • MSTN signals initially by binding to the activin type 2 receptors, ACVR2 and ACVR2B (12, 20-22), followed by engagement of the type 1 receptors, ALK4 and ALK5 (22, 23).
  • MSTN as a negative regulator of muscle growth is partially redundant with that of another TGF- ⁇ family member, activin A (20, 24-27), which shares many regulatory and signaling components with MSTN.
  • FST follicle stimulating hormone
  • FST was originally identified for its ability to inhibit secretion of follicle stimulating hormone (FSH) by cultured pituitary cells (28), and subsequent work showed that FST is capable of binding and inhibiting activins (29), which are capable of signaling to pituitary gonadotrophs to induce FSH secretion (30).
  • FST undergoes alternative splicing to generate two isoforms, the full-length FST315 and a carboxyl-terminal truncated FST288 (31).
  • FST3O3 is derived from proteolytic cleavage of the C-terminal domain. All of the FST isoforms contain a heparin binding domain that mediates binding to cell surface proteoglycans. The presence of the C-terminal acidic tail in FST315, however, appears to neutralize the basic residues present in the heparin binding domain, and as a result, FST315 binds poorly to proteoglycans and is the predominant form of FST in the circulation. FST288, which lacks the C-terminal 26 amino acid extension, tends to remain locally sequestered following secretion.
  • FST by binding and inhibiting both MSTN and activin A, plays an important role in regulating muscle growth.
  • transgenic overexpression of FST in skeletal muscle leads to muscle hypertrophy, consistent with inhibition of the MSTN/activin A signaling pathway (12), and conversely, heterozygous loss of Fst in mice leads not only to reductions in muscle weights (by about 15-20%), but also to a shift toward oxidative fiber types, an impairment of muscle regeneration following cardiotoxin-induced injury, and reduced tetanic force production (25), all consistent with overactivity of this signaling pathway.
  • Fst mice have also been shown to have a reduced amount of muscle at birth (32), but because mice completely lacking FST are not viable, mice carrying a conditional Fst flox allele (33) were used to target Fst in specific cell types and regions of the body in order to examine the effects of FST loss in tissues of adult mice. Even after extensive backcrossing of the flox allele onto a C57BL/6 background, mice carrying this allele (in the absence of ere recombinase) were heavier than wild-type mice, with total body weights of Fst flox/flox mice being increased by 13% and 19% in males and females, respectively. These differences in body weights reflected increased expression of Fst from the flox allele, likely resulting from retention of the neomycin selection cassette in the targeted locus during the construction of this mutant line (33).
  • Fst from thc flox allele allowed for the generation of mice carrying various combinations of wild-type, deletion, and flox alleles to produce an allelic series with varying levels of Fst expression.
  • Analysis of the gastrocnemius muscle showed that Fst RNA expression levels ranged from a 30% decrease in Fst +/- mice to 55% and 82% increases in Fst flox/+ and Fst flox/flox mice, respectively (FIG. 5A).
  • Levels of circulating FST also generally followed the same trends, with serum FST levels being approximately 50% and 200% of wild- type levels in Fst +/- and Fst flox/flox mice, respectively (FIG. 5B).
  • Fst expression levels correlated not only with total body weight (Table SI) but also with weights of individual muscles, which ranged from decreases of 18-23% in Fst +/- mice to increases of 29- 48% in Fst flox/flox mice depending on the specific muscle and sex (FIGs. 5A, 5C).
  • FsF lox/+ and Fst flox/- mice had intermediate muscle weights reflecting intermediate Fst expression levels in these mice.
  • FST acts in a dose-dependent manner to regulate muscle mass, with an approximately linear relationship between levels of FST expression and muscle weights.
  • mice carrying one or more Fst flox alleles in conjunction with Myl1 -Cre generally had lower circulating FST levels compared to mice lacking Myl1 -Cre, demonstrating that myofiber-derived FST does contribute to the circulating pool (FIG. 5B); similar trends were seen in female mice, although the individual comparisons were not statistically significant.
  • Targeting Fst in myofibers resulted in decreases in muscle weights in both males and females; although the effects were relatively small, many of the differences were statistically significant in both sexes (FIG. 5C).
  • myofiber-derived FST does play some role in regulating muscle mass.
  • Cdx2-cre transgene which is expressed specifically in the posterior but not anterior region of the animal (36); in particular, Cdx2-cre is expressed in all cells posterior to the umbilicus but not in any cells anterior to the umbilicus.
  • Fst +/- Cdx2-cre males were crossed with Fst flox/flox females, and FSF IOX/ ⁇ offspring that were either negative or positive for Cdx2-cre were analyzed.
  • Fst flox/- Cdx2-cre mice were viable, which allowed for analysis of mice at adulthood.
  • F66 transgenic mice which exhibit dramatic increases in muscle mass as a result of an Myl1 -Fst transgene located on the Y chromosome (24).
  • fiber number in the gastrocnemius was increased by 12% in F66 mice compared to wild-type mice, and the distribution of fiber diameters, which was slightly more spread out than in wild-type mice, was shifted to larger diameters, with mean fiber diameter being increased from 42.5 pm in wild-type mice to 59.7 ⁇ m in F66 mice (FIG. 5E).
  • Fst expression a 4-fold range in muscle size was generated, from a slightly over 50% decrease in Fst flox/- ; Cdx2-cre mice to an approximate doubling in F66 mice.
  • RNA- seq analysis identified 399 up-regulated and 234 down-regulated transcripts in gastrocnemius muscles isolated from Cdx2-cre positive compared to ere negative mice (FIG. 11).
  • these differentially regulated genes were ones encoding myosin heavy chain isoforms characteristic of specific fiber types (38, 39).
  • Myh7, Myh7B, and Myh2 were all up-regulated in Cdx2-cre positive muscles, consistent with increased numbers of type 1 and type 2a fibers, and Mhy4 was down-regulated, consistent with decreased numbers of type 2b fibers (FIG. 6B).
  • myosin light chain including myosin light chain, troponin, and tropomyosin isoforms
  • myosin light chain, troponin, and tropomyosin isoforms including myosin light chain, troponin, and tropomyosin isoforms, with components characteristic of slow fibers (Myl2, Myl3, Myl6b, Myl1O, Myl12a, Tnncl, Tnnil, Tnntl, and Tpm3) being up-regulated and components characteristic of fast fibers (Myl1, Mylpf, Tnnc2, Tnni2, Tnnt3, and TpmF) being down-regulated in Cdx2-cre positive muscles.
  • slow fibers Myl2, Myl3, Myl6b, Myl1O, Myl12a, Tnncl, Tnnil, Tnntl, and Tpm3
  • fast fibers Myl1, Mylpf, Tnnc2, Tnni2, Tnnt
  • myosin light chain kinase isoforms also tracked with these fiber type shifts, with Mylk3 (slow fibers) and Mylk2 (fast fibers) being up-regulated and down-regulated, respectively.
  • up-regulation of certain sarcomere protein isoforms not typically expressed in adult skeletal muscle, including Myh8 (neonatal), Myl4 (embryonic), Tnnt2 (cardiac), and Tpm2 (cardiac) were observed, raising the possibility that there might be enhanced regeneration occurring in Cdx2-cre positive muscles. However, no increase was observed in the number of centrally-located nuclei in muscles of Cdx2-cre positive mice.
  • RNA- seq analysis of F66 muscles with 2275 up-regulated and 2667 down-regulated transcripts compared to wild-type muscles (FIG. 11).
  • the down-regulated transcripts in F66 muscles were 75 that were oppositely regulated (i.e. up-regulated) in Cdx2-cre positive mice.
  • Pathway analysis of this subset of 75 genes identified three enriched pathways, namely thermogenesis, TCA cycle, and oxidative phosphorylation.
  • These three enriched pathways which comprised overlapping sets of genes, included a total of 17 genes whose functions were consistent with the shift toward oxidative fibers seen in Cdx2-cre positive mice and shift toward glycolytic fibers seen in F66 mice (Table 3).
  • up-regulated transcripts in F66 muscles were 101 that were oppositely regulated (down-regulated) in Cdx2-cre positive mice.
  • Pathway analysis of this subset of 101 genes identified seven enriched pathways, encompassing an overlapping set of 38 genes.
  • 38 genes were Rps6kbl encoding ribosomal protein S6 kinase B1, which plays an important role in regulating protein synthesis, three proto- oncogenes (Braf, Mras, Nras), and genes encoding 13 components of the cytoskeleton and extracellular matrix, including ankyrin 1, radixin, sarcoglycan alpha, decorin, thrombospondin 1, two integrin subunits, and six collagen chains (Table S3).
  • RNA-seq analysis of the gastrocnemius muscles identified a total of 633 differentially expressed transcripts between Cdx2-cre positive and ere negative mice
  • RNA-seq analysis of triceps muscles revealed no differences between Cdx2-cre positive compared to ere negative mice with an adjusted p value less than 0.05.
  • FST acts predominantly, if not exclusively, in a local manner to regulate muscle size, fiber type, and lipid accumulation.
  • FST is capable of blocking the activities of ligands signaling through activin type 2 receptors
  • FIGs. 8A-8B micro-CT analysis of femurs, humeri, and L4 and L5 vertebrae showed opposite trends in Fst flox/flox and Fst +/- mice in comparison to wild-type mice.
  • parameters such as bone volume, bone surface, BV/TV, connectivity density, trabecular number, trabecular thickness, and bone mineral density were generally higher in bones of Fst flox/flox mice and were generally lower in bones of Fst +/ ' mice compared to those of wild-type mice.
  • mice carrying floxed alleles for Alk4 and Alk5 were used to examine the effect of simultaneously targeting these type 1 receptors in osteoblasts utilizing the Oc-cre transgene.
  • mice in which Acvr2 and Acvr2b were simultaneously targeted using the same Oc-cre transgene were also analyzed.
  • BV/TV was increased by 12-13-fold and bone mineral density was increased by 8-9-fold in humeri, femurs, and L5 vertebrae of Alk4 flox/flox ; Alk5 flox/flox ; Oc-cre mice compared to ere negative mice.
  • targeting signaling specifically in osteoblasts leads to massive increases in bone volume and density, with the effects of targeting the two type 1 receptors being much more pronounced compared to targeting the two type 2 receptors.
  • targeting signaling in osteoblasts is sufficient to cause changes in bone structure, including increases in bone mineral density and density.
  • the effects on bone structure and density were extensive upon targeting the two type 1 receptors, ALK4 and ALK5.
  • the increases in parameters such as BV/TV and bone mineral density seen upon targeting ALK4 and ALK5 were quite remarkable, reaching levels of 12-13 fold in the case of BV/TV and 8-9 fold in the case of bone mineral density.
  • TGF- ⁇ is known to utilize ALK5 for signaling, but TGF- ⁇ utilizes a different type 2 receptor, namely TGFBR2, to couple to ALK5.
  • Fiber diameters were measured (as the shortest distance across each fiber passing through the midpoint) from hematoxylin and eosin-stained sections. Measurements were carried out on 250 fibers per muscle, and all data for a given genotype were pooled. Fiber type analysis was carried out using antibodies (BA-D5, SC-71, and BF-F3 for myosin heavy chains type I, Ila, and lib, respectively) developed by Schiaffino el al.
  • GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne des méthodes d'inhibition de la signalisation du récepteur de type 1 et/ou du récepteur de type 2 chez des sujets, éventuellement des sujets présentant des maladies associées à la signalisation de la myostatine, du récepteur de type 1 et/ou du récepteur de type 2, ou à la perte musculaire et/ou à la détérioration osseuse. L'invention concerne également des compositions d'inhibition de la signalisation du récepteur de type 1 et/ou du récepteur de type 2.
PCT/US2021/059157 2020-11-13 2021-11-12 Agents thérapeutiques ciblant la signalisation de la famille bêta du facteur de croissance transformant WO2022104071A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US18/036,704 US20230414711A1 (en) 2020-11-13 2021-11-12 Therapeutics targeting transforming growth factor beta family signaling
EP21892877.8A EP4243933A2 (fr) 2020-11-13 2021-11-12 Agents thérapeutiques ciblant la signalisation de la famille bêta du facteur de croissance transformant

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063113575P 2020-11-13 2020-11-13
US63/113,575 2020-11-13
US202163227232P 2021-07-29 2021-07-29
US63/227,232 2021-07-29

Publications (3)

Publication Number Publication Date
WO2022104071A2 true WO2022104071A2 (fr) 2022-05-19
WO2022104071A3 WO2022104071A3 (fr) 2022-06-23
WO2022104071A9 WO2022104071A9 (fr) 2022-10-13

Family

ID=81602616

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/059157 WO2022104071A2 (fr) 2020-11-13 2021-11-12 Agents thérapeutiques ciblant la signalisation de la famille bêta du facteur de croissance transformant

Country Status (3)

Country Link
US (1) US20230414711A1 (fr)
EP (1) EP4243933A2 (fr)
WO (1) WO2022104071A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1771171A1 (fr) * 2004-07-29 2007-04-11 Schering-Plough Ltd. Utilisation d'inhibiteurs des recepteurs alk5 pour moduler ou inhiber l'activite de la myostatine entrainant une accretion de tissus maigres chez des animaux
CA2831827A1 (fr) * 2011-04-05 2012-10-11 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Composes et procedes pour la modification de la signalisation par kinase du type du recepteur d'activine
MA41919A (fr) * 2015-04-06 2018-02-13 Acceleron Pharma Inc Hétéromultimères alk4:actriib et leurs utilisations
CN111867632A (zh) * 2018-03-23 2020-10-30 赛途公司 骨骼肌肥大诱导剂的alk5抑制剂

Also Published As

Publication number Publication date
US20230414711A1 (en) 2023-12-28
WO2022104071A9 (fr) 2022-10-13
WO2022104071A3 (fr) 2022-06-23
EP4243933A2 (fr) 2023-09-20

Similar Documents

Publication Publication Date Title
Tomlinson et al. NGF-TrkA signaling by sensory nerves coordinates the vascularization and ossification of developing endochondral bone
Sartori et al. BMP signaling controls muscle mass
Lories et al. Articular cartilage and biomechanical properties of the long bones in Frzb‐knockout mice
Rodino‐Klapac et al. Inhibition of myostatin with emphasis on follistatin as a therapy for muscle disease
Rossi et al. Nfix regulates temporal progression of muscle regeneration through modulation of myostatin expression
Mavalli et al. Distinct growth hormone receptor signaling modes regulate skeletal muscle development and insulin sensitivity in mice
Harish et al. Inhibition of myostatin improves muscle atrophy in oculopharyngeal muscular dystrophy (OPMD)
Lee et al. Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A
Squarzoni et al. Interleukin‐6 neutralization ameliorates symptoms in prematurely aged mice
Guo et al. Prevascularized scaffolds bearing human dental pulp stem cells for treating complete spinal cord injury
Lahoute et al. Premature aging in skeletal muscle lacking serum response factor
Lee et al. NPY signalling in early osteoblasts controls glucose homeostasis
Sartoretto et al. Apoptosis‐associated speck‐like protein containing a caspase‐1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis
US10953028B2 (en) Method of treating heterotopic ossification
Maurizi et al. RNA interference therapy for autosomal dominant osteopetrosis type 2. Towards the preclinical development
Magadum et al. Therapeutic Delivery of Pip4k2c‐Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart
Shao et al. Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality
Maupin et al. Enhanced cortical bone expansion in Lgals3-deficient mice during aging
US20230414711A1 (en) Therapeutics targeting transforming growth factor beta family signaling
US20090304625A1 (en) Modulators of cardiac cell hypertrophy and hyperplasia
KR101890874B1 (ko) 근육 노화 억제용 또는 근육 노화 관련 질환 치료용 조성물
Fahrner et al. microRNA-501 controls myogenin+/CD74+ myogenic progenitor cells during muscle regeneration
Zanotti et al. The Dmp1‐SOST Transgene Interacts With and Downregulates the Dmp1‐Cre Transgene and the RosaNotch Allele
Gomez et al. Contrasting effects of Ksr2, an obesity gene, on trabecular bone volume and bone marrow adiposity
US20150306185A1 (en) Heparanase and its uses related to exostoses

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21892877

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021892877

Country of ref document: EP

Effective date: 20230613

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21892877

Country of ref document: EP

Kind code of ref document: A2